HLA-A24 agonist epitopes of MUC1-C oncoprotein and compositions and methods of use

ABSTRACT

The invention provides a human cytotoxic T lymphocyte (CTL) agonist epitope from the C-terminal subunit of mucin 1 (MUC1-C), which can be used as a peptide, polypeptide (protein), and/or in vaccine or other composition for the prevention or therapy of cancer. The invention further provides a nucleic acid encoding the peptide, protein, or polypeptide, a vector comprising the nucleic acid, a cell comprising the peptide, polypeptide, nucleic acid, or vector, and compositions thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional application of U.S. Ser. No.16/715,038, filed Dec. 16, 2019, which is a is a divisional applicationof U.S. Ser. No. 16/034,654, filed Jul. 13, 2018, now U.S. Pat. No.10,508,141, which is a divisional application of U.S. Ser. No.15/031,435, filed Apr. 22, 2016, now U.S. Pat. No. 10,035,832, which isthe U.S. national phase of PCT/US2014/061723, filed Oct. 22, 2014, whichclaims the benefit of U.S. Provisional Patent Application No.61/894,482, filed Oct. 23, 2013, all of which are incorporated herein byreference.

REFERENCE TO SEQUENCE LISTING

This application contains a Sequence Listing submitted electronically asa text file by EFS-Web. The text file, named “723806_ST25”, has a sizein bytes of 58,000 Bytes, and was recorded on Apr. 22, 2016. Theinformation contained in the text file is incorporated herein byreference in its entirety pursuant to 37 CFR § 1.52(e)(5)

BACKGROUND OF THE INVENTION

MUC1 (CD227) is a type I membrane glycoprotein composed of heterodimersof a large N-terminal subunit (MUC1-N) covalently bound to a smallC-terminal subunit (MUC1-C).

The N-terminal subunit (MUC1-N) is the large extracellular domain, whichconsists of the variable number of tandem repeats region (VNTR) and thenon-VNTR region. MUC1-N is shed from the cells and can be found in thecirculation of patients with advanced cancer. MUC1-N is used as a tumormarker (CA15.3) in breast cancer patients (see Hayes et al., J. Clin.Oncol., 4: 1542-50 (1986)).

The C-terminal region of MUC1 (MUC1-C) has three distinctive parts: asmall extracellular domain that is covalently bound to MUC1-N, a singletransmembrane domain, and a cytoplasmic tail (see Lan et al., J. Biol.Chem., 265: 15294-9 (1990)). The cytoplasmic tail contains sites forinteraction with signaling proteins, such as β-catenin, epidermal growthfactor receptor (EGFR), and Src (see Li et al., J. Biol. Chem., 276:35239-42 (2001)). Since these proteins are situated at the basolateralpart of healthy cells, protein-MUC1 interactions are not believed to besignificant. However, loss of polarity in human tumor cells allows thecytoplasmic tail to be exposed to the signaling proteins, andinteraction can occur (see Vermeer et al., Nature, 422: 322-6 (2003)).

The MUC1-C region has been shown to act as an oncogene, leading totransformation of human cells when MUC1-C binds to β-catenin (see Li etal., Oncogene, 22: 6107-10 (2003); Raina et al., Cancer Res., 69:5133-41 (2009); and Wei et al., Cancer Res., 67: 1853-8 (2007)).Moreover, MUC1-C transfection has been demonstrated to be sufficient toinduce transformation and confer oncogenic activities previouslyattributed to the full-length MUC1 protein, such as increased growthrate, anchorage-independent cell growth, and resistance to chemotherapyagents (see Ren et al., Cancer Cell. 5: 163-75 (2004)). In addition,MUC1-C signaling activated by c-Src is involved in the disruption ofboth E-cadherin adherens junctions and integrin focal adhesions thatstimulate cancer cell motility, invasion, and metastasis, therebysuggesting a possible role for MUC1-C in epithelial to mesenchymaltransition (EMT) (see Hu et al., Expert Rev. Anticancer Ther., 6:1261-71 (2006)). Overexpression of genes related to MUC1 has also beenfound to be highly associated with poor prognosis in patients with lungand breast cancer and with drug resistance (see Ren et al., supra; andKhodarev et al., Cancer Res., 69: 2833-7 (2009)).

Numerous clinical trials have evaluated MUC1 as a potential target forvaccine therapy of a range of human tumors. The majority of these haveemployed polypeptides of the VNTR region. One agonist epitope (P93L) wasshown, compared to the native epitope, to enhance the generation of Tcells that can also more efficiently lyse human tumor cells (see Tsanget al., Cancer Res., 10: 2139-49 (2004)). Two other potential agonistepitopes in this region were shown to enhance T-cell cytokineproduction, but no tumor cell killing was reported (see Mitchell et al.,Cancer Immunol. Immunother., 56: 287-301 (2007)).

One method that has been shown to enhance the ability of a vaccine to bemore efficacious is to make alterations in the amino acid sequence ofputative T-cell epitopes, which in turn can enhance T-cell activationand specific T-cell killing of tumor cells (see Grey et al., CancerSurv., 22: 37-49 (1995); and Terasawa et al., Clin. Cancer Res. 8: 41-53(2002)). Not all substitutions of an amino acid of a potential cytotoxicT lymphocyte (CTL) epitope, however, will lead to an enhancer agonistepitope, and some substitutions will lead to antagonist epitopes.Moreover, the generation of a putative agonist epitope of a tumorassociated antigen may well lead to enhanced T-cell activation by IFN-γproduction, but will be useless unless the activated T cell willrecognize the endogenous (native) epitope expressed in the context ofthe MHC on the surface of human tumor cells, and consequently lyse thosetumor cells.

There is a desire to identify new specific cytotoxic T lymphocyte (CTL)epitopes and enhancer agonist peptides or epitopes of MUC1-C, and todevelop compositions and methods that use these epitopes for thediagnosis and/or treatment of cancer.

BRIEF SUMMARY OF THE INVENTION

The invention provides a peptide comprising the amino acid sequence ofSEQ ID NO: 1 or SEQ ID NO: 2.

In another aspect, the invention provides a polypeptide (protein)comprising the peptide, a nucleic acid encoding the peptide, a vectorcomprising the nucleic acid, a cell comprising the peptide, polypeptide(protein), nucleic acid, or vector, and compositions thereof.

In particular, the invention provides a MUC1 protein or polypeptidecomprising the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2(e.g., SEQ ID NO: 16).

In another aspect, the invention provides a yeast-MUC1 immunotherapeuticcomposition comprising (a) a yeast vehicle and (b) a fusion proteincomprising at least one MUC1 antigen, wherein the MUC1 antigen comprisesthe amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The invention also provides a yeast-MUC1 immunotherapeutic compositioncomprising (a) a yeast vehicle and (b) a fusion protein comprising atleast one MUC1 antigen, wherein the MUC1 antigen comprises an amino acidsequence that is at least 80% identical to (i) SEQ ID NO: 16, (ii)positions 92-566 of SEQ ID NO: 16, or (iii) a corresponding sequencefrom a different MUC1 protein, and wherein the MUC1 antigen comprisesthe amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

The invention also provides a yeast-MUC1 immunotherapeutic compositioncomprising (a) a yeast vehicle and (b) a fusion protein comprising atleast one MUC1 antigen, wherein the MUC1 antigen comprises an amino acidsequence that differs from an amino acid sequence of a wild-type MUC1protein by at least one amino acid substitution at a sequence position,with respect to a wild-type MUC1 amino acid sequence such as SEQ ID NO:14, that is selected from: T422, P430, T431, 5462, and A470.

The invention also provides a method of enhancing an immune responseagainst a MUC1-expressing cancer in a host comprising administering atherapeutically effective amount of a composition comprising thepeptide, protein, polynucleotide, nucleic acid, vector, cell, oryeast-MUC1 immunotherapeutic composition to the host, wherein the immuneresponse in the host is enhanced.

The invention also provides a method of treating a MUC1-expressingcancer in an individual comprising administering a therapeuticallyeffective amount of a composition comprising the peptide, polypeptide(protein), nucleic acid, vector, cell, or yeast-MUC1 immunotherapeuticcomposition to the individual.

The invention also provides a method of reducing, arresting, reversingor preventing the metastatic progression of cancer in an individual whohas a MUC1-expressing cancer comprising administering a therapeuticallyeffective amount of a composition comprising the peptide, polypeptide(protein), nucleic acid, vector, cell, or yeast-MUC1 immunotherapeuticcomposition to the individual.

The invention also provides a method of preventing or delaying the onsetof a MUC1-expressing cancer in an individual comprising administering atherapeutically effective amount of a composition comprising thepeptide, polypeptide (protein), nucleic acid, vector, cell, oryeast-MUC1 immunotherapeutic composition to the individual.

The invention further provides a method of inhibiting a MUC1-expressingcancer in a subject comprising (a) obtaining (isolating) lymphocytesfrom the subject, (b) stimulating the lymphocytes with a compositioncomprising the peptide, polypeptide (protein), nucleic acid, vector, orcell to the host to generate cytotoxic T lymphocytes ex vivo, and (c)administering the cytotoxic T lymphocytes to the subject, wherein theMUC1-expressing cancer in the subject is inhibited.

The invention provides a method of inhibiting a MUC1-expressing cancerin a subject comprising (a) obtaining (isolating) dendritic cells fromthe subject, (b) treating the dendritic cells with a compositioncomprising the peptide, polypeptide (protein), nucleic acid, vector,cell, or yeast-MUC1 immunotherapeutic composition ex vivo, and (c)administering the treated dendritic cells to the subject, wherein theMUC1-expressing cancer in the subject is inhibited.

Additionally, the invention provides inhibiting a MUC1-expressing cancerin a subject comprising (a) obtaining peripheral blood mononuclear cells(PBMCs) from a subject suffering from cancer, (b) isolating dendriticcells from the PBMCs, (c) treating the dendritic cells with acomposition comprising the peptide, polypeptide (protein), nucleic acid,vector, cell, or yeast-MUC1 immunotherapeutic composition ex vivo, (d)activating the PBMCs with the treated dendritic cells ex vivo, and (e)administering the activated PBMCs to the subject, wherein theMUC1-expressing cancer in the subject is inhibited.

The invention further provides inhibiting a MUC1-expressing cancer in asubject comprising (a) obtaining peripheral blood mononuclear cells(PBMCs) from a subject suffering from cancer, (b) isolating dendriticcells from the PBMCs, (c) treating the dendritic cells with acomposition comprising the peptide, polypeptide (protein), nucleic acid,vector, cell or yeast-MUC1 immunotherapeutic composition ex vivo, (d)activating the PBMCs with the treated dendritic cells ex vivo, (e)isolating T lymphocytes from the activated PBMCs ex vivo, and (f)administering the isolated T lymphocytes to the subject, wherein theMUC1-expressing cancer in the subject is inhibited.

The invention provides the use of adoptively transferred T cellsstimulated in vitro with a composition comprising the peptide,polypeptide (protein), nucleic acid, vector, cell, or yeast-MUC1immunotherapeutic composition to treat a cancer, to inhibit aMUC1-expressing cancer in a subject, to reduce, arrest, reverse, orprevent the metastatic progression of cancer in an individual that hascancer, or to prevent or delay the onset of a MUC1-expressing cancer.

In an additional aspect, the invention provides a method of inducing animmune response against a MUC1-expressing cancer in a subject comprising(a) administering to the subject a first poxviral vector comprising anucleic acid encoding the amino acid sequence of SEQ ID NO: 1 or SEQ IDNO: 2 and (b) administering to the subject a second poxviral vectorcomprising a nucleic acid encoding the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 2. In one embodiment, the nucleic acid encoding theamino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is a nucleic acidencoding a MUC1 protein comprising the amino acid sequence of SEQ ID NO:1 or SEQ ID NO: 2 (e.g., SEQ ID NO: 16).

DETAILED DESCRIPTION OF THE INVENTION

The invention provides peptides comprising a human cytotoxic Tlymphocyte (CTL) epitope from the C-terminal subunit of humantumor-associated antigen (TAA) mucin 1 (MUC1) and analogs thereof, whichcan be used in vaccines and other compositions for the prevention ortherapeutic treatment of cancer, including, but not limited to, a cancerthat expresses or overexpresses MUC1. In particular, the inventionprovides peptides, polypeptides, and proteins comprising, consistingessentially of, or consisting of the amino acid sequence of KYHPMSEYAL(SEQ ID NO: 1) or KYTNPAVAL (SEQ ID NO: 2).

In another embodiment, the invention provides a polypeptide thatcomprises the MUC1 amino acid sequence (i.e., a MUC1 protein) orfragment thereof, wherein one or more of the corresponding amino acidresidues have been replaced with one or more of the enhancer agonistepitopes SEQ ID NO: 1 or SEQ ID NO: 2 (e.g., SEQ ID NO: 16).

A “polypeptide” is generally understood to be a linear organic polymerconsisting of a large number of amino acid residues bonded together in acontinuous, unbranched chain, forming part of, or the whole of, aprotein molecule. A “peptide” is generally considered to bedistinguished from a full-length protein or polypeptide on the basis ofsize, and, in one embodiment, as an arbitrary benchmark can beunderstood to contain approximately 50 or fewer amino acids, whilepolypeptides or full-length proteins are generally longer. However, theterms “peptide” and “polypeptide” can be used interchangeably in someembodiments to describe a protein useful in the present invention, orthe term “protein” can be used generally.

The inventive peptide or polypeptide can be any suitable length. In oneembodiment, a peptide of the invention has no more than 20 (e.g., nomore than 19, no more than 18, no more than 17, no more than 16, no morethan 15, no more than 14, no more than 13, no more than 12, no more than11, or no more than 10) amino acid residues. The additional amino acidresidues, if present, preferably are from the MUC1 (e.g., MUC1-C)protein or based on the sequence of MUC1 as described herein. Theadditional amino acid residues can be positioned at either end or bothends of the amino acid sequence of SEQ ID NO: 1 or SEQ ID NO: 2.

A polypeptide for expression in a host cell, such as a yeast, is of aminimum size capable of being expressed recombinantly in the host cell.Accordingly, the polypeptide that is expressed by the host cell ispreferably at least 25 amino acids in length, and is typically at leastor greater than 25 amino acids in length, or at least or greater than 26amino acids, at least or greater than 27 amino acids, at least orgreater than 28 amino acids, at least or greater than 29 amino acids, atleast or greater than 30 amino acids, at least or greater than 31 aminoacids, at least or greater than 32 amino acids, at least or greater than33 amino acids, at least or greater than 34 amino acids, at least orgreater than 35 amino acids, at least or greater than 36 amino acids, atleast or greater than 37 amino acids, at least or greater than 38 aminoacids, at least or greater than 39 amino acids, at least or greater than40 amino acids, at least or greater than 41 amino acids, at least orgreater than 42 amino acids, at least or greater than 43 amino acids, atleast or greater than 44 amino acids, at least or greater than 45 aminoacids, at least or greater than 46 amino acids, at least or greater than47 amino acids, at least or greater than 48 amino acids, at least orgreater than 49 amino acids, or at least or greater than 50 amino acidsin length, or at least 25-50 amino acids in length, at least 30-50 aminoacids in length, or at least 35-50 amino acids in length, or at least40-50 amino acids in length, or at least 45-50 amino acids in length,although smaller proteins may be expressed, and considerably largerproteins (e.g., hundreds of amino acids in length or even a few thousandamino acids in length) may be expressed.

In another embodiment, the invention provides a polypeptide which can beused in vaccines and other compositions for the prevention ortherapeutic treatment of cancer, including but not limited to cancersthat express or overexpress MUC1, wherein the polypeptide comprises,consists essentially of, or consists of a MUC1 amino acid sequence orfragment thereof (e.g., an immunogenic domain thereof), wherein one ormore of the corresponding amino acid residues of the polypeptide havebeen replaced (e.g., substituted) such that the polypeptide comprisesone or more of the enhancer agonist epitopes of SEQ ID NO: 1 or SEQ IDNO: 2 (i.e., the polypeptide has an amino acid sequence that differsfrom a native, or wild-type, MUC1 amino acid sequence in that the aminoacid sequence of the polypeptide comprises one or more of the enhanceragonist epitopes, which typically involves the substitution of one, two,three or more amino acids in a given wild-type epitope sequence with adifferent amino acid). In one aspect of this embodiment, the polypeptidecan further comprise additional MUC1 enhancer agonist epitopes, examplesof which are described in detail below.

Peptides and polypeptides (proteins) of the invention are, in someembodiments of the invention, used as antigens. According to the presentinvention, the general use herein of the term “antigen” refers to anyportion of a protein (e.g., peptide, partial protein, full-lengthprotein), wherein the protein is naturally occurring or syntheticallyderived or designed, to a cellular composition (whole cell, cell lysateor disrupted cells), to an organism (whole organism, lysate or disruptedcells), or to a carbohydrate, or other molecule, or a portion thereof.An antigen may elicit an antigen-specific immune response (e.g., ahumoral and/or a cell-mediated immune response) against the same orsimilar antigens that are encountered in vitro, in vivo, or ex vivo byan element of the immune system (e.g., T cells, antibodies).

An antigen can be as small as a single epitope (e.g., SEQ ID NO: 1 orSEQ ID NO: 2 described herein), a single immunogenic domain or larger,and can include multiple epitopes or immunogenic domains. As such, thesize of a protein antigen can be as small as about 8-11 amino acids(e.g., a peptide) and as large as a domain of a protein, a full-lengthprotein, a multimer, a fusion protein, or a chimeric protein. Antigensuseful in various immunotherapeutic compositions described hereininclude peptides, polypeptides, protein domain(s) (e.g., immunogenicdomains), protein subunits, full-length proteins, multimers, fusionproteins, and chimeric proteins.

When referring to stimulation of an immune response, the term“immunogen” is a subset of the term “antigen” and, therefore, in someinstances, can be used interchangeably with the term “antigen.” Animmunogen, as used herein, describes an antigen which elicits a humoraland/or cell-mediated immune response (i.e., is immunogenic), such thatadministration of the immunogen to an individual mounts anantigen-specific immune response against the same or similar antigensthat are encountered by the immune system of the individual. In oneembodiment, the immunogen elicits a cell-mediated immune response,including a CD4⁺ T cell response (e.g., TH1, TH2, and/or TH17) and/or aCD8⁺ T cell response (e.g., a CTL response).

An “immunogenic domain” or “immunological domain” of a given protein(polypeptide) can be any portion, fragment or epitope of an antigen(e.g., a peptide fragment or subunit or an antibody epitope or otherconformational epitope) that contains at least one epitope that can actas an immunogen when administered to an animal. Therefore, animmunogenic domain is larger than a single amino acid and is at least ofa size sufficient to contain at least one epitope that can act as animmunogen. For example, a single protein can contain multiple differentimmunogenic domains. Immunogenic domains need not be linear sequenceswithin a protein, such as in the case of a humoral immune response,where conformational domains are contemplated.

An epitope is defined herein as a single immunogenic site within a givenantigen that is sufficient to elicit an immune response when provided tothe immune system in the context of appropriate costimulatory signalsand/or activated cells of the immune system. In other words, an epitopeis the part of an antigen that is recognized by components of the immunesystem, and may also be referred to as an antigenic determinant. Thoseof skill in the art will recognize that T cell epitopes are different insize and composition from B cell or antibody epitopes, and that epitopespresented through the Class I MHC pathway differ in size and structuralattributes from epitopes presented through the Class II MHC pathway. Forexample, T cell epitopes presented by Class I MHC molecules aretypically between 8 and 11 amino acids in length, whereas epitopespresented by Class II MHC molecules are less restricted in length andmay be up to 25 amino acids or longer. In addition, T cell epitopes havepredicted structural characteristics depending on the specific MHCmolecules bound by the epitope. Epitopes can be linear sequence epitopesor conformational epitopes (conserved binding regions). Most antibodiesrecognize conformational epitopes.

A “target antigen” is an antigen that is specifically targeted by animmunotherapeutic composition of the invention (i.e., an antigen,usually the native antigen, against which elicitation of an immuneresponse is desired, even if the antigen used in the immunotherapeuticis an agonist of the native antigen). A “cancer antigen,” which also isreferred to as a tumor-associated antigen (TAA), is an antigen thatcomprises at least one antigen that is associated with a cancer, such asan antigen expressed by a tumor cell, so that targeting the antigen alsotargets the tumor cell and/or cancer. A cancer antigen can include oneor more antigens from one or more proteins, including one or moretumor-associated proteins. In particular, a “MUC1 antigen” is an antigenthat is derived, designed, or produced from a MUC1 protein (includingMUC1-N, MUC1-C or both MUC1-N and MUC1-C). A “MUC1 agonist antigen” isan antigen derived, designed, or produced from a MUC1 protein (includingMUC1-N, MUC1-C or both MUC1-N and MUC1-C) that includes at least oneagonist epitope, such as the enhancer agonist epitopes described herein.Preferred enhancer agonist epitopes of the invention have an amino acidsequence of SEQ ID NO: 1 or SEQ ID NO: 2.

MUC1 (which may also be referred to as “mucin-1,” “DF3 antigen,” or“HMFG1”) is a large glycoprotein expressed by most epithelial secretorytissues at basal levels and is expressed at high levels by malignanciesof epithelial cell origin. MUC1 is most typically found as apolymorphic, type I transmembrane protein with a large extracellulardomain (also referred to as MUC1-N subunit) that includes variablenumbers of tandem repeats (VNTR; typically between 20 and 125 repeats)that are highly glycosylated through 0-linkages. The MUC1 protein isencoded as a single transcript, and then processed into subunitspost-translationally, known as MUC1-N and MUC1-C, or α and β subunits,respectively, which then form a heterodimeric protein by a strongnoncovalent interaction of the two subunits. MUC1 is cleaved into its N-and C-subunits within the “sea urchin sperm protein, enterokinase andagrin” (SEA) domain, a highly conserved protein domain that was namedbased on its initial identification in a sperm protein, in enterokinase,and in agrin, and that is found in a number of heavily glycosylatedmucin-like proteins that are typically membrane-tethered. The MUC1protein is cleaved between glycine and serine residues present in thesequence GSVVV, which corresponds to positions 1097-1101 of SEQ ID NO:11, within the SEA domain (Lillehoj et al., Biochem. Biophys. Res.Commun., 307: 743-749 (2003); Parry et al., Biochem. Biophys. Res.Commun., 283: 715-720 (2001); Wreschner et al., Protein Sci., 11:698-706 (2002)).

The MUC1-C subunit includes the extracellular domain (ED), which isglycosylated and binds the galectin-3 ligand, which in turn serves as abridge to physically associate MUC1 with the epidermal growth factorreceptor (EGFR) and possibly other receptor tyrosine kinases. MUC1-Calso comprises a transmembrane (TM) domain, and a cytoplasmic domain(CD) which contains several tyrosine residues which, whenphosphorylated, could act as binding motifs for proteins with SH2domains (for a detailed discussion of the MUC1 protein and known andputative functions, see Kufe, Cancer Biol. & Ther., 7: 81-84 (2008)).Alternative splice variants of MUC1 (known as MUC1/Y and MUC1/X, forexample) are “short” versions of MUC1 that lack most of MUC1-N,including the large VNTR region, but that include the ED, TM and CDregions, as well as the SEA domain and portions of the N-terminal regionsignal sequence region. Cleavage within the SEA domain may not occur inthese short versions.

The isolation and sequencing of DNA and cDNA encoding human MUC1 hasbeen reported (see, e.g., Siddiqui et al., PNAS, 85: 2320-2323 (1998);Abe and Kufe, PNAS, 90: 282-286 (1993); Hareuveni et al., Eur. J.Biochem., 189(3): 475-486 (1990); Gendler et al., J. Biol. Chem.,265(25): 15286-15293 (1990); Lan et al., J. Biol. Chem., 265(25):15294-15299 (1990); Tsarfaty et al., Gene, 93(2): 313-318 (1990);Lancaster, Biochem. Biophys. Res. Commun., 173(3): 1019-1029 (1990)). Anexample of a full-length human MUC1 precursor protein containing boththe MUC1-N and MUC1-C regions is described in SwissProt Accession No.P15941.3 (GI:296439295), and is represented here by SEQ ID NO: 5. 10different MUC1 isoforms can be created from the gene encoding SEQ ID NO:5 by alternative transcript splicing. For example, an isoform known asMUC1/Y lacks positions 54-1053 of SEQ ID NO: 5. Various other isoformsare described in the database description of this protein.

A variety of transcript variants of MUC1 are known, but the MUC1subunits, domains, or regions described in the exemplary SEQ ID NO: 5above can readily be identified in the variants, such that a MUC1antigen useful in the invention can be designed or produced based on agiven MUC1 sequence, or a corresponding sequence from another MUC1protein. For example, one nucleotide sequence encoding a human MUC1protein is represented herein by SEQ ID NO: 6, which corresponds toGENBANK® Accession No. NM_002456.4 (GI: 65301116). SEQ ID NO: 6 encodesa 273 amino acid human MUC1 protein (transcript variant 1, also known asMUC1/ZD), the amino acid sequence of which is represented here as SEQ IDNO: 7 (also found in GENBANK® Accession No. NP_002447.4; GI:65301117).Another nucleotide sequence encoding another human MUC1 protein isrepresented herein by SEQ ID NO: 8, which corresponds to GENBANK®Accession No. NM_001018016.1 (GI:67189006). SEQ ID NO: 8 encodes a 264amino acid human MUC1 protein (transcript variant 2, also known as“MUC1/Y”), the amino acid sequence of which is represented here as SEQID NO: 9 (also found in GENBANK® Accession No. NP_001018016.1;GI:67189007). Another nucleotide sequence encoding another human MUC1protein is represented herein by SEQ ID NO: 10, which corresponds toGENBANK® Accession No. AY327587.1 (GI:33150003). SEQ ID NO: 10 encodes a264 amino acid human MUC1 protein (transcript variant 2, also known as“MUC1/Y”), the amino acid sequence of which is represented here as SEQID NO: 11 (also found in GENBANK® Accession No. AAP97018.1 (GI:33150004). Another nucleotide sequence encoding another human MUC1protein is represented herein by SEQ ID NO: 12, which corresponds toGENBANK® Accession No. NM_001018017 (GI:324120954). SEQ ID NO: 12encodes a 255 amino acid human MUC1 protein (transcript variant 3), theamino acid sequence of which is represented here as SEQ ID NO: 13 (alsofound in GENBANK® Accession No. NP_001018017.1; GI:67189069). Yetanother exemplary wild-type MUC1 amino acid sequence is represented hereby SEQ ID NO: 14 (also found in GENBANK® Accession No. NP_001191214).SEQ ID NO: 14 is used as a reference for some of the amino acidpositions of MUC1 described herein, but the corresponding positions inother MUC1 sequences can be identified by those of skill in the art.

Human MUC1, including the human MUC1 proteins and MUC1 antigensdescribed herein, contains various CD4⁺ and CD8⁺ T cell epitopes. Such Tcell epitopes have been described, for example, in U.S. Pat. Nos.6,546,643; 7,118,738; 7,342,094; 7,696,306; and U.S. Patent ApplicationPublication No. 2008/0063653, as well as in PCT Publication No. WO2013/024972, and any one or more of these epitopes can be used in a MUC1antigen of the present invention, including by adding, deleting orsubstituting one or more amino acids within a sequence described hereinto conform the sequence to the published epitope sequence at thatposition(s).

Examples of MUC1 agonist antigens discovered in the present inventionare provided herein (see Examples). A peptide, protein, or polypeptideuseful in the present invention comprises, consists essentially of, orconsists of at least one of the MUC1 enhancer agonist peptidesrepresented by SEQ ID NO: 1 and SEQ ID NO: 2. However, other MUC1agonist epitopes can be additionally included in a MUC1 antigen for usein the present invention. In one embodiment, a MUC1 agonist antigensuitable for use in the present invention comprises a MUC1 protein orpolypeptide or peptide thereof having an amino acid sequence thatdiffers from the wild-type (native) MUC1 protein or polypeptide orpeptide thereof by at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, or more amino acid substitutions, where the amino acidsubstitutions introduce one or more MUC1 agonist epitopes into theantigen. Such amino acid substitutions can include substitutions at thefollowing amino acid positions, where the positions of the substitutionsare provided with respect to a wild-type MUC1 having an amino acidsequence represented by Accession No. NP 001191214 (SEQ ID NO: 14)(although the same or equivalent positions can be readily identified inany other wild-type MUC1 sequence): T93, A141, P142, G149, S150, T151,A392, C406, T422, P430, T431, T444, D445, S460, S462, and/or A470. Inone embodiment, the substitution is: T93L, A141Y, P142L, G149V, S150Y,T151L, A392Y, C406V, T422K, P430A, T431L, T444L, D445F, S460Y, S462K,and/or A470L.

In addition, a MUC1 antigen useful in the present invention may includeone or more additional amino acid mutations (substitutions, insertionsor deletions), for example, to inactivate or delete a natural biologicalfunction of the native protein (e.g., to improve expression or enhancesafety of the antigen). One example of such a mutation is aninactivating mutation that is a substitution at position C404 withrespect to the wild-type protein using SEQ ID NO: 14 as a referencesequence. In one aspect, the inactivating substitution is C404A (withrespect to SEQ ID NO: 14).

The peptide or polypeptide (protein) of the invention can be prepared byany method, such as by synthesizing the peptide or by expressing anucleic acid encoding an appropriate amino acid sequence for the peptideor polypeptide in a cell and, in some embodiments, harvesting thepeptide or polypeptide from the cell. In some embodiments, the peptideor polypeptide is not harvested from the cell, such as in embodiments ofthe invention directed to a yeast-based immunotherapy composition, whichis described in detail below. A combination of such methods ofproduction of peptides and polypeptides also can be used. Methods of denovo synthesizing peptides and methods of recombinantly producingpeptides or polypeptides are known in the art (see, e.g., Chan et al.,Fmoc Solid Phase Peptide Synthesis, Oxford University Press, Oxford,United Kingdom, 2005; Peptide and Protein Drug Analysis, ed. Reid, R.,Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., OxfordUniversity Press, Oxford, United Kingdom, 2000; Sambrook et al.,Molecular Cloning: A Laboratory Manual, 3^(rd) ed., Cold Spring HarborPress, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates and JohnWiley & Sons, N Y, 1994).

The invention also provides a nucleic acid molecule comprising a nucleicacid sequence encoding the peptide or the polypeptide. The nucleic acidmolecule can comprise DNA (genomic or cDNA) or RNA, and can be single ordouble stranded. Furthermore, the nucleic acid molecule can comprisenucleotide analogues or derivatives (e.g., inosine or phophorothioatenucleotides and the like). The nucleic acid sequence can encode thepeptide or polypeptide alone or as part of a fusion protein. The nucleicacid sequence encoding the peptide or polypeptide can be provided aspart of a construct comprising the nucleic acid molecule and elementsthat enable delivery of the nucleic acid molecule to a cell, and/orexpression of the nucleic acid molecule in a cell. Such elementsinclude, for example, expression vectors, promoters, and transcriptionand/or translation control sequences. Such constructs can also bereferred to as “recombinant nucleic acid molecules”. Suitable vectors,promoters, transcription/translation sequences, and other elements, aswell as methods of preparing such nucleic acid molecules and constructs,are known in the art (e.g., Sambrook et al., supra; and Ausubel et al.,supra). Although the phrase “nucleic acid molecule” primarily refers tothe physical nucleic acid molecule and the phrase “nucleic acidsequence” primarily refers to the sequence of nucleotides on the nucleicacid molecule, the two phrases can be used interchangeably, especiallywith respect to a nucleic acid molecule, or a nucleic acid sequence,being capable of encoding a peptide or polypeptide. Similarly, thephrase “recombinant nucleic acid molecule” primarily refers to a nucleicacid molecule operatively linked to an element such as a transcriptioncontrol sequence, but can be used interchangeably with the phrase“nucleic acid molecule.”

The invention further provides a vector comprising the nucleic acidmolecule. Examples of suitable vectors include plasmids (e.g., DNAplasmids) and viral vectors, such as poxvirus, retrovirus, adenovirus,adeno-associated virus, herpes virus, polio virus, alphavirus,baculorvirus, and Sindbis virus.

In a first embodiment, the vector is a plasmid (e.g., DNA plasmid). Theplasmid can be complexed with chitosan.

In a second embodiment, the vector is a poxvirus (e.g., chordopox virusvectors and entomopox virus vectors). Suitable poxviruses includeorthopox, avipox, parapox, yatapox, and molluscipox, raccoon pox, rabbitpox, capripox (e.g., sheep pox), leporipox, and suipox (e.g., swinepox).Examples of avipox viruses include fowlpox, pigeonpox, canarypox, suchas ALVAC, mynahpox, uncopox, quailpox, peacockpox, penguinpox,sparrowpox, starlingpox, and turkeypox. Examples of orthopox virusesinclude smallpox (also known as variola), cowpox, monkeypox, vaccinia,ectromelia, camelpox, raccoonpox, and derivatives thereof.

The term “vaccinia virus” refers to both the wild-type vaccinia virusand any of the various attenuated strains or isolates subsequentlyisolated including, for example, modified vaccinia Ankara (MVA), NYVAC,TROYVAC, Dry-Vax (also known as vaccinia virus-Wyeth), PDXVAC-TC(Schering-Plough Corporation), vaccinia virus-Western Reserve, vacciniavirus-EM63, vaccinia virus-Lister, vaccinia virus-New York City Board ofHealth, vaccinia virus-Temple of Heaven, vaccinia virus-Copenhagen,ACAM1000, ACAM2000, and modified vaccinia virus Ankara-Bavarian Nordic(“MVA-BN”).

In certain embodiments, the MVA is selected from the group consisting ofMVA-572, deposited at the European Collection of Animal Cell Cultures(“ECACC”), Health Protection Agency, Microbiology Services, Porton Down,Salisbury SP4 0JG, United Kingdom (“UK”), under the deposit number ECACC94012707 on Jan. 27, 1994; MVA-575, deposited at the ECACC under depositnumber ECACC 00120707 on Dec. 7, 2000; MVA-Bavarian Nordic (“MVA-BN”),deposited at the ECACC under deposit number V00080038 on Aug. 30, 2000;and derivatives of MVA-BN. Additional exemplary poxvirus vectors aredescribed in U.S. Pat. No. 7,211,432.

The vaccinia virus MVA was generated by 516 serial passages on chickenembryo fibroblasts of the Ankara strain of Vaccinia virus, referred toas chorioallantois virus Ankara (CVA) (see Mayr et al., Infection, 3:6-14 (1975)). The genome of the resulting attenuated MVA lacksapproximately 31 kilobase pairs of genomic DNA compared to the parentalCVA strain and is highly host-cell restricted to avian cells (see Meyeret al., J. Gen. Virol., 72: 1031-1038 (1991)). It was shown in a varietyof animal models that the resulting MVA was significantly avirulent(Mayr et al., Dev. Biol. Stand., 41: 225-34 (1978)). This MVA strain hasbeen tested in clinical trials as a vaccine to immunize against smallpoxin humans (see Mary et al., Zbl. Bakt. Hyg. I, Abt. Org. B, 167: 375-390(1987); and Stickl et al., Dtsch. Med. Wschr., 99: 2386-2392 (1974)).Those studies involved over 120,000 humans, including high-riskpatients, and proved that compared to vaccinia virus-based vaccines, MVAhad diminished virulence or infectiousness while still able to induce agood specific immune response. Although MVA-BN is preferred for itsbetter safety profile because it is less replication competent thanother MVA strains, all MVAs are suitable for this invention, includingMVA-BN and its derivatives.

Both MVA and MVA-BN are able to efficiently replicate their DNA inmammalian cells even though they are avirulent. This trait is the resultof losing two important host range genes among at least 25 additionalmutations and deletions that occurred during its passages throughchicken embryo fibroblasts (see Meyer et al., Gen. Virol., 72: 1031-1038(1991); and Antoine et al., Virol., 244: 365-396 (1998)). In contrast tothe attenuated Copenhagen strain (NYVAC) and host range restrictedavipox (ALVAC), both-early and late transcription in MVA are unimpaired,which allows for continuous gene expression throughout the viral lifecycle (see Sutter et al., Proc. Nat'l Acad. Sci. USA, 89:10847-10851(1992)). In addition, MVA can be used in conditions of pre-existingpoxvirus immunity (Ramirez et al., J. Virol., 74: 7651-7655 (2000)).

Both MVA and MVA-BN lack approximately 15% (31 kb from six regions) ofthe genome compared with the ancestral chorioallantois vaccinia virusAnkara (“CVA”). The deletions affect a number of virulence and hostrange genes, as well as the gene for Type A inclusion bodies. MVA-BN canattach to and enter human cells where virally-encoded genes areexpressed very efficiently. However, assembly and release of progenyvirus does not occur. MVA-BN is strongly adapted to primary chickenembryo fibroblast (CEF) cells and does not replicate in human cells. Inhuman cells, viral genes are expressed, and no infectious virus isproduced. Despite its high attenuation and reduced virulence, inpreclinical studies, MVA-BN has been shown to elicit both humoral andcellular immune responses to vaccinia and to heterologous gene productsencoded by genes cloned into the MVA genome (see Harrer et al., Antivir.Ther., 10(2): 285-300 (2005); Cosma et al., Vaccine, 22(1): 21-29(2003); Di Nicola et al., Hum. Gene Ther., 14(14): 1347-1360 (2003); andDi Nicola et al., Clin. Cancer Res., 10(16): 5381-5390 (2004)).

The reproductive replication of a virus is typically expressed by theamplification ratio. The term “amplification ratio” refers to the ratioof virus produced from an infected cell (“output”) to the amountoriginally used to infect the cells in the first place (“input”). Anamplification ratio of “1” defines an amplification status in which theamount of virus produced from infected cells is the same as the amountinitially used to infect the cells, which means that the infected cellsare permissive for virus infection and reproduction. An amplificationratio of less than 1 means that infected cells produce less virus thanthe amount used to infect the cells in the first place, and indicatesthat the virus lacks the capability of reproductive replication, whichis a measure of virus attenuation.

Thus, the term “not capable of reproductive replication” means that anMVA or MVA derivative has an amplification ratio of less than 1 in oneor more human cell lines, such as, for example, the human embryonickidney 293 cell line (HEK293, which is deposited under deposit numberECACC No. 85120602), the human bone osteosarcoma cell line 143B(deposited under deposit number ECACC No. 91112502), the human cervixadenocarcinoma cell line HeLa (deposited at the American Type CultureCollection (ATTC) under deposit number ATCC No. CCL-2), and the humankeratinocyte cell line HaCat (see Boukamp et al., J. Cell Biol., 106(3):761-71 (1988)).

MVA-BN does not reproductively replicate in the human cell lines HEK293,143B, HeLa, and HaCat (see U.S. Pat. Nos. 6,761,893 and 6,193,752, andInternational Patent Application Publication No. WO 2002/042480). Forexample, in one exemplary experiment, MVA-BN exhibited an amplificationratio of 0.05 to 0.2 in HEK293 cells, an amplification ratio of 0.0 to0.6 in 143B cells, an amplification ratio of 0.04 to 0.8 in HeLa cells,and an amplification ratio of 0.02 to 0.8 in HaCat cells. Thus, MVA-BNdoes not reproductively replicate in any of the human cell lines HEK293,143B, HeLa, and HaCat. In contrast, the amplification ratio of MVA-BN isgreater than 1 in primary cultures of chicken embryo fibroblast cells(CEF) and in baby hamster kidney cells (BHK, which is deposited underdeposit number ATCC No. CRL-1632). Therefore MVA-BN can easily bepropagated and amplified in CEF primary cultures with an amplificationratio above 500, and in BHK cells with an amplification ratio above 50.

As noted above, all MVAs are suitable for this invention, includingMVA-BN and its derivatives. The term “derivatives” refers to virusesshowing essentially the same replication characteristics as the straindeposited with ECACC on Aug. 30, 2000, under deposit number ECACC No.V00080038 but showing differences in one or more parts of its genome.Viruses having the same “replication characteristics” as the depositedvirus are viruses that replicate with similar amplification ratios asthe deposited strain in CEF cells, in BHK cells, and in the human celllines HEK293, 143B, HeLa, and HaCat.

When the vector is for administration to a host (e.g., human), thevector (e.g., poxvirus) preferably has a low replicative efficiency in atarget cell (e.g., no more than about 1 progeny per cell or, morepreferably, no more than 0.1 progeny per cell are produced). Replicationefficiency can readily be determined empirically by determining thevirus titer after infection of the target cell.

In addition to the nucleic acid molecule encoding the polypeptide(protein) or polypeptide (i.e., the peptide or polypeptide comprising,consisting essentially of, or consisting of at least one MUC1 enhanceragonist epitope described herein), a vector useful in the invention(e.g., a plasmid or a viral vector) also can comprise a nucleic acidsequence encoding one or more immunostimulatory/regulatory molecules,granulocyte macrophage colony stimulating factor (GM-CSF), cytokines,and/or molecules that can enhance an immune response (e.g., additionaltumor-associated antigens). Exemplary additional tumor-associatedantigens (TAAs, also referred to as cancer antigens) include, but arenot limited to, 5-α-reductase, α-fetoprotein (AFP), AM-1, APC, April, Bmelanoma antigen gene (BAGE), β-catenin, Bcl12, bcr-abl, Brachyury,CA-125, caspase-8 (CASP-8 also known as FLICE), Cathepsins, CD19, CD20,CD21/complement receptor 2 (CR2), CD22/BL-CAM, CD23/F_(c)εRII, CD33,CD35/complement receptor 1 (CR1), CD44/PGP-1, CD45/leucocyte commonantigen (LCA), CD46/membrane cofactor protein (MCP), CD52/CAMPATH-1,CD55/decay accelerating factor (DAF), CD59/protectin, CDC27, CDK4,carcinoembryonic antigen (CEA), c-myc, cyclooxygenase-2 (cox-2), deletedin colorectal cancer gene (DCC), DcR3, E6/E7, CGFR, EMBP, Dna78,farnesyl transferase, fibroblast growth factor-8a (FGF8a), fibroblastgrowth factor-8b (FGF8b), FLK-1/KDR, folic acid receptor, G250, Gmelanoma antigen gene family (GAGE-family), gastrin 17,gastrin-releasing hormone, ganglioside 2 (GD2)/ganglioside 3(GD3)/ganglioside-monosialic acid-2 (GM2), gonadotropin releasinghormone (GnRH), UDP-GlcNAc:R₁Man(α1-6)R₂ [GlcNAc to Man(α1-6)]β1,6-N-acetylglucosaminyltransferase V (GnT V), GP1, gp100/Pme117,gp-100-in4, gp15, gp75/tyrosine-related protein-1 (gp75/TRP-1), humanchorionic gonadotropin (hCG), heparanase, Her2/neu, human mammary tumorvirus (HMTV), 70 kiloDalton heat-shock protein (HSP70), human telomerasereverse transcriptase (hTERT), insulin-like growth factor receptor-1(IGFR-1), interleukin-13 receptor (IL-13R), inducible nitric oxidesynthase (iNOS), Ki67, KIAA0205, K-ras, H-ras, N-ras, KSA, LKLR-FUT,melanoma antigen-encoding family (MAGE-family, including at leastMAGE-1, MAGE-2, MAGE-3, and MAGE-4), mammaglobin, MAP17,Melan-A/melanoma antigen recognized by T-cells-1 (MART-1), mesothelin,MIC A/B, MT-MMPs, mucin, testes-specific antigen NY-ESO-1, osteonectin,p15, P170/MDR1, p53, p97/melanotransferrin, PAI-1, platelet-derivedgrowth factor (PDGF), μPA, PRAME, probasin, progenipoietin,prostate-specific antigen (PSA), prostate-specific membrane antigen(PSMA), RAGE-1, Rb, RCAS1, mutated Ras, SART-1, SSX-family, STAT3, STn,TAG-72, transforming growth factor-alpha (TGF-α), transforming growthfactor-beta (TGF-β), Thymosin-beta-15, tumor necrosis factor-alpha(TNF-α), TP1, TRP-2, tyrosinase, vascular endothelial growth factor(VEGF), ZAG, p16INK4, and glutathione-S-transferase (GST), as well asmodified versions thereof (e.g., CEA-6D).

In the case of a viral vector, the nucleic acid encoding the peptide, aswell as any other exogenous gene(s), preferably are inserted into a siteor region (insertion region) in the vector (e.g., poxvirus) that doesnot affect virus viability of the resultant recombinant virus. Suchregions can be readily identified by testing segments of virus DNA forregions that allow recombinant formation without seriously affectingvirus viability of the recombinant virus.

The thymidine kinase (TK) gene is an insertion region that can readilybe used and is present in many viruses. In particular, the TK gene hasbeen found in all examined poxvirus genomes. Additional suitableinsertion sites are described in International Patent ApplicationPublication WO 2005/048957. For example, in fowlpox, insertion regionsinclude, but are not limited to, the BamHI J fragment, EcoRI-HindIIIfragment, BamHI fragment, EcoRV-HindIII fragment, long unique sequence(LUS) insertion sites (e.g., FPV006/FPV007 and FPV254/FPV255), FP14insertion site (FPV060/FPV061), and 43K insertion site (FPV107/FPV108).In vaccinia, insertion sites include, but are not limited to, 44/45,49/50, and 124/125.

When the vector is a recombinant fowlpox virus comprising a nucleic acidencoding the peptide and/or other exogenous gene(s) (e.g., encoding oneor more immunostimulatory/regulatory molecules), the nucleic acidencoding the peptide can be inserted in one region (e.g., the FP14region), and the exogenous gene(s) can be inserted in another region(e.g., the BamHI J region).

The inventive vector can include suitable promoters and regulatoryelements, such as a transcriptional regulatory element or an enhancer.Suitable promoters include the SV40 early promoter, an RSV promoter, theretrovirus LTR, the adenovirus major late promoter, the human CMVimmediate early I promoter, and various poxvirus promoters, such as thePr7.5K promoter, 30K promoter, 40K promoter, I3 promoter, Prs promoter,PrsSynIIm promoter, PrLE1 promoter, synthetic early/late (sE/L)promoter, HH promoter, 11K promoter, and Pi promoter. While thepromoters typically will be constitutive promoters, inducible promotersalso can be used in the inventive vectors. Such inducible systems allowregulation of gene expression.

In one embodiment of the invention, a cell comprising (1) the peptide orpolypeptide, (2) a nucleic acid molecule encoding the peptide orpolypeptide, and/or (3) a vector comprising the nucleic acid moleculealso is provided herein. Suitable cells include prokaryotic andeukaryotic cells, e.g., mammalian cells, yeast, fungi other than yeast,and bacteria (such as E. coli). The cell can be used in vitro, such asfor research or for production of the peptide or polypeptide, or thecell can be used in vivo. In one embodiment, the cell is a yeast cell,which may be used to provide a yeast vehicle component of theyeast-based immunotherapy composition as described herein. In anotherembodiment, the cell can be a peptide-pulsed antigen presenting cell.Suitable antigen presenting cells include, but are not limited to,dendritic cells, B lymphocytes, monocytes, macrophages, and the like.

In one embodiment, the cell is dendritic cell. Dendritic cells ofdifferent maturation stages can be isolated based on the cell surfaceexpression markers. For example, mature dendritic cells are less able tocapture new proteins for presentation but are much better at stimulatingresting T cells to grow and differentiate. Thus, mature dendritic cellscan be of importance. Mature dendritic cells can be identified by theirchange in morphology and by the presence of various markers. Suchmarkers include, but are not limited to, cell surface markers such asB7.1, B7.2, CD40, CD11, CD83, and MHC class II. Alternatively,maturation can be identified by observing or measuring the production ofpro-inflammatory cytokines.

Dendritic cells can be collected and analyzed using typicalcytofluorography and cell sorting techniques and devices, such as afluorescence-activated cell sorter (FACS). Antibodies specific to cellsurface antigens of different stages of dendritic cell maturation arecommercially available.

In one embodiment, the cell is a yeast (e.g., Saccharomyces).Accordingly, the invention also provides a yeast-based immunotherapeuticcomposition comprising (a) a yeast vehicle and (b) an antigen comprisinga MUC1 peptide or polypeptide (protein) of the invention (also generallyreferred to herein as “yeast-immunotherapy composition,”“yeast-immunotherapy product,” “yeast-immunotherapeutic composition,”“yeast-based vaccine,” or derivatives of these phrases). A yeast-basedimmunotherapeutic composition that contains a MUC1 antigen can bereferred to more specifically as a “yeast-MUC1 immunotherapeuticcomposition” or derivatives thereof as noted above. An“immunotherapeutic composition” is a composition that elicits an immuneresponse sufficient to achieve at least one therapeutic benefit in asubject. A “yeast-based immunotherapeutic composition” (and derivativesthereof) refers to a composition that includes a yeast vehicle componentand an antigen component, and can elicit or induce an immune response,such as a cellular immune response, including without limitation a Tcell-mediated cellular immune response. The immune response generallyincludes both an innate immune response and an adaptive immune response,and is generated against both the yeast component and the antigencomponent (an antigen-specific immune response). Preferably, theyeast-based immunotherapeutic composition, when administered to anindividual, provides at least one protective, preventative, ortherapeutic benefit to the individual. In one aspect, a yeast-basedimmunotherapeutic composition useful in the invention is capable ofinducing a CD8⁺ and/or a CD4⁺ T cell-mediated immune response and in oneaspect, a CD8⁺ and a CD4⁺ T cell-mediated immune response, particularlyagainst a target antigen (e.g., a cancer antigen, and preferably againstMUC1). A CD4⁺ immune response can include TH1 immune responses, TH2immune responses, TH17 immune responses, or any combination of theabove. A CD8⁺ immune response can include a cytotoxic T lymphocyte (CTL)response. In one aspect, a yeast-based immunotherapeutic compositionmodulates the number and/or functionality of regulatory T cells (Tregs)in a subject.

As described above, a yeast-based immunotherapy composition of theinvention includes (a) a yeast vehicle and (b) at least one cancerantigen comprising a MUC1 antigen or immunogenic domain thereof, wherethe MUC1 antigen comprises, consists essentially of, or consists of, atleast one MUC1 enhancer agonist epitope having an amino acid sequenceselected from SEQ ID NO: 1 and/or SEQ ID NO: 2. The cancer antigen isexpressed by (i.e., recombinantly), attached to, loaded into, or mixedwith the yeast vehicle.

In some embodiments, the cancer antigen, MUC1 antigen, or immunogenicdomain thereof is provided as a fusion protein. For example, severalMUC1 proteins and fusion proteins have been described in PCT PublicationNo. WO 2013/024972. Such proteins and fusion proteins can be furthermodified to incorporate the enhancer agonist epitopes of the presentinvention. In some embodiments, the cancer antigen and the MUC1 antigenare the same element. In some embodiments, the cancer antigen includesother antigens, including other cancer antigens (also referred to hereinas tumor associated antigens or TAAs) in addition to the MUC1 antigen.In one aspect of the invention, a fusion protein useful as a cancerantigen can include two or more antigens, e.g., a MUC1 antigen andanother cancer antigen (TAA) that is not a MUC1 antigen, or twodifferent MUC1 antigens. In one aspect, the fusion protein can includetwo or more immunogenic domains of one or more antigens, such as two ormore immunogenic domains of a MUC1 antigen, or two or more epitopes ofone or more antigens, such as two or more epitopes of a MUC1 antigen. Avariety of other cancer antigens or TAAs are known in the art and aredescribed elsewhere herein.

An example of a MUC1 antigen that is useful in an inventive yeast-basedimmunotherapy composition comprises or consists of the amino acidsequence of SEQ ID NO: 16. SEQ ID NO: 16 is the amino acid sequence of afusion protein comprising a MUC1 antigen for use in a yeast-basedimmunotherapy composition, where the MUC1 antigen is a full-length MUC1agonist protein corresponding to a wild-type MUC1 protein except for (a)the introduction of 15 amino acid substitutions to form several agonistepitopes within the protein, including the enhancer agonist epitope ofSEQ ID NO: 1 and (b) a single amino acid substitution that is aninactivating mutation. SEQ ID NO: 16 includes the following sequences inthe following order from N- to C-terminus: (1) an alpha factor leadersequence of SEQ ID NO:17 (corresponding to positions 1-89 of SEQ ID NO:16); (2) a linker sequence of Thr-Ser to facilitate cloning(corresponding to positions 90-91 of SEQ ID NO: 16); (3) a full-lengthMUC1 agonist protein corresponding to a wild-type protein except for theintroduction of the above-mentioned 15 amino acid agonist substitutionsand one inactivating substitution (corresponding to positions 92-566 ofSEQ ID NO: 16) and (4) a hexapeptide histidine tag (corresponding topositions 567-572 of SEQ ID NO: 16).

SEQ ID NO: 16 is encoded by the nucleotide sequence represented by SEQID NO: 15 (codon optimized for yeast expression). The alpha leadersequence (corresponding to positions 1-89 of SEQ ID NO: 16) could besubstituted with a different N-terminal sequence designed to impartresistance to proteasomal degradation and/or stabilize expression, suchas the peptide represented by SEQ ID NO: 19 or an N-terminal peptidefrom a different yeast alpha leader sequence, such as SEQ ID NO: 18, orby a MUC1 signal sequence. The hexahistidine C-terminal tag is optionaland facilitates identification and/or purification of the protein.

As compared to the wild-type MUC1 protein used as a template, thesequence of SEQ ID NO: 16 contains the following amino acidsubstitutions: (substitution positions given with reference to SEQ IDNO: 16 with further reference in parentheses to the location of thesubstitution in a wild-type MUC1 represented by Accession No. NP001191214 corresponding to SEQ ID NO: 14): T184L (position 93 inwild-type MUC1), A232Y (position 141 in wild-type MUC1), P233L (position142 in wild-type MUC1), G240V (position 149 in wild-type MUC1), S241Y(position 150 in wild-type MUC1), T242L (position 151 in wild-typeMUC1), A483Y (position 392 in wild-type MUC1), C495A (position 404 inwild-type MUC1) C497V (position 406 in wild-type MUC1), T513K (position422 in wild-type MUC1), P521A (position 430 in wild-type MUC1), T522L(position 431 in wild-type MUC1), T535L (position 444 in wild-typeMUC1), D536F (position 445 in wild-type MUC1), and S551Y (position 460in wild-type MUC1). The substitution C495A (position 404 in thewild-type MUC1 protein) is the inactivating mutation; the remainder ofthe substitutions are to produce agonist epitopes.

SEQ ID NO: 16 comprises the enhancer agonist peptide referred to hereinas SEQ ID NO: 1. SEQ ID NO: 1 is located at positions 513-522 of SEQ IDNO: 16.

The MUC1 antigen for yeast-based immunotherapy represented by SEQ ID NO:16 contains agonist epitopes for several different HLA types, includingA2, A3 and A24, making it a versatile and unique antigen for targetingtumors in a variety of individuals with a MUC1 expressing cancer.

A MUC1 antigen useful in the yeast-based immunotherapy composition ofthe present invention also includes antigens having an amino acidsequence that is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical tothe amino acid sequence of SEQ ID NO: 16 over the full length of thefusion protein or over a defined fragment of SEQ ID NO: 16 (e.g., animmunological domain or functional domain (domain with at least onebiological activity)) that forms part of the protein, including, but notlimited to, positions 92-566 of SEQ ID NO: 16 (the MUC1 antigen withinSEQ ID NO: 16).

It is straightforward to use the corresponding portions of any of theMUC1 proteins that are derived or obtained from sequence or sourcesother than those exemplified herein, and particularly from sequences orsources within the same animal species, to create peptides,polypeptides, and fusion proteins having a similar or the same overallstructure as the peptides, polypeptides, and fusion proteins describedherein. By way of example, one can readily identify a correspondingsequence in a given human MUC1 protein from any source that correspondsto positions 92-566 of SEQ ID NO: 16 using simple sequence alignmenttools or processes. Therefore, sequences with minor and/or conservativedifferences from the sequences exemplified herein are expresslyencompassed by the present invention.

As discussed above, N-terminal expression sequences and the C-terminaltags, such as those described above with respect to the fusion proteinof SEQ ID NO: 16 are optional, but may be selected from severaldifferent sequences to improve or assist with expression, stability,and/or allow for identification and/or purification of the protein. Forexample, an exemplary N-terminal synthetic sequence that enhances thestability of expression of an antigen in a yeast cell and/or preventspost-translational modification of the protein in the yeast includes thesequence M-A-D-E-A-P (represented herein by SEQ ID NO: 19). In otherembodiments, the MUC1 antigen is linked at the N-terminus to a yeastprotein, such as an alpha factor prepro sequence (also referred to asthe alpha factor signal leader sequence, the amino acid sequence ofwhich is exemplified herein by SEQ ID NO: 17 or SEQ ID NO: 18). Othersequences for yeast alpha factor prepro sequence are known in the artand are encompassed for use in the present invention. Also, manydifferent promoters suitable for use in yeast are known in the art.Furthermore, short intervening linker sequences (e.g., 1, 2, 3, 4, or 5amino acid peptides) may be introduced between portions of a fusionprotein comprising a MUC1 antigen for a variety of reasons, includingthe introduction of restriction enzyme sites to facilitate cloning, ascleavage sites for host phagosomal proteases, to accelerate protein orantigen processing, and for future manipulation of the constructs.

For use in embodiments of the invention directed to yeast, any suitableyeast promoter can be used and a variety of such promoters are known tothose skilled in the art. Promoters for expression in Saccharomycescerevisiae include, but are not limited to, promoters of genes encodingthe following yeast proteins: alcohol dehydrogenase I (ADH1) or II(ADH2), CUP1, phosphoglycerate kinase (PGK), triose phosphate isomerase(TPI), translational elongation factor EF-1 alpha (TEF2),glyceraldehyde-3-phosphate dehydrogenase (GAPDH; also referred to asTDH3, for triose phosphate dehydrogenase), galactokinase (GAL1),galactose-1-phosphate uridyl-transferase (GAL7), UDP-galactose epimerase(GAL10), cytochrome c1 (CYC1), Sec7 protein (SECT) and acid phosphatase(PHOS), including hybrid promoters such as ADH2/GAPDH and CYC1/GAL10promoters, and including the ADH2/GAPDH promoter, which is induced whenglucose concentrations in the cell are low (e.g., about 0.1 to about 0.2percent), as well as the CUP1 promoter and the TEF2 promoter. Likewise,a number of upstream activation sequences (UASs), also referred to asenhancers, are known. Upstream activation sequences for expression inSaccharomyces cerevisiae include, but are not limited to, the UASs ofgenes encoding the following proteins: PCK1, TPI, TDH3, CYC1, ADH1,ADH2, SUC2, GAL1, GAL7 and GAL10, as well as other UASs activated by theGAL4 gene product, with the ADH2 UAS being used in one aspect. Since theADH2 UAS is activated by the ADR1 gene product, it may be preferable tooverexpress the ADR1 gene when a heterologous gene is operatively linkedto the ADH2 UAS. Transcription termination sequences for expression inSaccharomyces cerevisiae include the termination sequences of theα-factor, GAPDH, and CYC1 genes.

Transcription control sequences to express genes in methyltrophic yeastinclude the transcription control regions of the genes encoding alcoholoxidase and formate dehydrogenase.

According to the present invention, a “yeast vehicle” used in ayeast-based immunotherapy composition is any yeast cell (e.g., a wholeor intact cell) or a derivative thereof (see below) that can be used inconjunction with one or more antigens, immunogenic domains thereof, orepitopes thereof in a yeast-based immunotherapeutic composition of theinvention (e.g., a therapeutic or prophylactic composition). The yeastvehicle therefore can include, but is not limited to, a live intact(whole) yeast microorganism (i.e., a yeast cell having all itscomponents including a cell wall), a killed (dead) or inactivated intactyeast microorganism, derivatives of intact yeast including a yeastspheroplast (i.e., a yeast cell lacking a cell wall), a yeast cytoplast(i.e., a yeast cell lacking a cell wall and nucleus), a yeast ghost(i.e., a yeast cell lacking a cell wall, nucleus, and cytoplasm), asubcellular yeast membrane extract or fraction thereof (also referred toas a yeast membrane particle and previously as a subcellular yeastparticle), any other yeast particle, or a yeast cell wall preparation.

Yeast spheroplasts are typically produced by enzymatic digestion of theyeast cell wall. Such a method is described, for example, in Franzusoffet al., Meth. Enzymol., 194: 662-674 (1991). Yeast cytoplasts aretypically produced by enucleation of yeast cells. Such a method isdescribed, for example, in Coon, Natl. Cancer Inst. Monogr., 48: 45-55(1978). Yeast ghosts are typically produced by resealing a permeabilizedor lysed cell and can, but need not, contain at least some of theorganelles of that cell. Such a method is described, for example, inFranzusoff et al., J. Biol. Chem., 258, 3608-3614 (1983) and Bussey etal., Biochim. Biophys. Acta, 553: 185-196 (1979). A yeast membraneparticle (subcellular yeast membrane extract or fraction thereof) refersto a yeast membrane that lacks a natural nucleus or cytoplasm. Theparticle can be of any size, including sizes ranging from the size of anatural yeast membrane to microparticles produced by sonication or othermembrane disruption methods known to those skilled in the art, followedby resealing. A method for producing subcellular yeast membrane extractsis described, for example, in Franzusoff et al., Meth. Enzymol., 194,662-674 (1991). One also can use fractions of yeast membrane particlesthat contain yeast membrane portions and, when the antigen or otherprotein is expressed recombinantly by the yeast prior to preparation ofthe yeast membrane particles, the antigen or other protein of interest.Antigens or other proteins of interest can be carried inside themembrane, on either surface of the membrane, or combinations thereof(i.e., the protein can be both inside and outside the membrane and/orspanning the membrane of the yeast membrane particle). In oneembodiment, a yeast membrane particle is a recombinant yeast membraneparticle that can be an intact, disrupted, or disrupted and resealedyeast membrane that includes at least one desired antigen or otherprotein of interest on the surface of the membrane or at least partiallyembedded within the membrane. An example of a yeast cell wallpreparation is a preparation of isolated yeast cell walls carrying anantigen on its surface or at least partially embedded within the cellwall such that the yeast cell wall preparation, when administered to asubject, stimulates a desired immune response against a disease target.

Any yeast strain can be used to produce a yeast vehicle of the presentinvention, or otherwise used as a host cell in the present invention.Yeast are unicellular microorganisms that belong to one of threeclasses: Ascomycetes, Basidiomycetes and Fungi Imperfecti. Oneconsideration for the selection of a type of yeast for use as an immunemodulator is the pathogenicity of the yeast. In one embodiment, theyeast is a non-pathogenic strain such as Saccharomyces cerevisiae. Theselection of a non-pathogenic yeast strain minimizes any adverse effectsto the individual to whom the yeast vehicle is administered. However,pathogenic yeast may be used if the pathogenicity of the yeast can benegated by any means known to one of skill in the art (e.g., mutantstrains). In accordance with one aspect of the present invention,non-pathogenic yeast strains are used.

Genera of yeast strains that may be used in the invention include butare not limited to Saccharomyces, Candida (which can be pathogenic),Cryptococcus, Hansenula, Kluyveromyces, Pichia, Rhodotorula,Schizosaccharomyces and Yarrowia. In one aspect, yeast genera areselected from Saccharomyces, Candida, Hansenula, Pichia orSchizosaccharomyces, and in one aspect, Saccharomyces is used. Speciesof yeast strains that may be used in the invention include but are notlimited to Saccharomyces cerevisiae, Saccharomyces carlsbergensis,Candida albicans, Candida kefyr, Candida tropicalis, Cryptococcuslaurentii, Cryptococcus neoformans, Hansenula anomala, Hansenulapolymorpha, Kluyveromyces fragilis, Kluyveromyces lactis, Kluyveromycesmarxianus var. lactis, Pichia pastoris, Rhodotorula rubra,Schizosaccharomyces pombe, and Yarrowia hpolytica. It is to beappreciated that a number of these species include a variety ofsubspecies, types, subtypes, etc. that are intended to be includedwithin the aforementioned species. In one aspect, yeast species used inthe invention include S. cerevisiae, C. albicans, H. polymorpha, P.pastoris and S. pombe. S. cerevisiae is useful as it is relatively easyto manipulate and being “Generally Recognized As Safe” or “GRAS” for useas food additives (GRAS, FDA proposed Rule 62FR18938, Apr. 17, 1997).One embodiment of the present invention is a yeast strain that iscapable of replicating plasmids to a particularly high copy number, suchas a S. cerevisiae cir° strain. The S. cerevisiae strain is one suchstrain that is capable of supporting expression vectors that allow oneor more target antigen(s) and/or antigen fusion protein(s) and/or otherproteins to be expressed at high levels. Another yeast strain is usefulin the invention is Saccharomyces cerevisiae W303α. In addition, anymutant yeast strains can be used in the present invention, includingthose that exhibit reduced post-translational modifications of expressedtarget antigens or other proteins, such as mutations in the enzymes thatextend N-linked glycosylation. In one aspect of the invention, ayeast-based immunotherapy composition is produced using a mutant yeaststrain that produces the MUC1 antigen as an underglycosylated protein ascompared to the same antigen produced by the wild-type strain (withnormal glycosylation). Such a MUC1 antigen may be more similar to MUC1antigens expressed by tumor cells, which can then be processed intounique T cell epitopes by antigen presenting cells, thus enhancing thespecific anti-tumor response.

In general, the yeast vehicle and antigen(s) and/or other agents can beassociated by any technique described herein. In one aspect, the yeastvehicle is loaded intracellularly with the antigen(s) and/or other oradditional agent(s) to be included in the composition. In anotheraspect, the antigen(s) and/or agent(s) is covalently or non-covalentlyattached to the yeast vehicle. In yet another aspect, the yeast vehicleand the antigen(s) and/or agent(s) are associated by mixing. In anotheraspect, the antigen(s) and/or agent(s) are expressed recombinantly bythe yeast vehicle or by the yeast cell or yeast spheroplast from whichthe yeast vehicle is derived (if the yeast vehicle is other than a wholeintact cell or a spheroplast).

In one embodiment, a yeast cell used to prepare the yeast vehicle istransfected with a heterologous nucleic acid molecule encoding a peptideor polypeptide (e.g., the antigen) such that the peptide or polypeptideis expressed by the yeast cell. Such a yeast also is referred to hereinas a recombinant yeast or a recombinant yeast vehicle. The yeast cellcan then be formulated with a pharmaceutically acceptable excipient andadministered directly to an individual, stored for later administrationto an individual, or loaded into a dendritic cell, which can then inturn be administered to an individual. The yeast cell also can bekilled, or it can be derivatized such as by formation of yeastspheroplasts, cytoplasts, ghosts, or subcellular particles, any of whichmay be followed by storing, administering directly to an individual, orloading of the cell or derivative into a dendritic cell. Yeastspheroplasts can also be directly transfected with a recombinant nucleicacid molecule (e.g., the spheroplast is produced from a whole yeast, andthen transfected) in order to produce a recombinant spheroplast thatexpresses the antigen. Yeast cells or yeast spheroplasts thatrecombinantly express the antigen(s) may be used to produce a yeastvehicle comprising a yeast cytoplast, a yeast ghost, or a yeast membraneparticle or yeast cell wall particle, or fraction thereof.

A number of antigens and/or other proteins to be produced by a yeastvehicle of the present invention is any number of antigens and/or otherproteins that can be reasonably produced by a yeast vehicle, andtypically ranges from at least one to at least about 6 or more,including from about 2 to about 6 antigens and or other proteins.

Expression of an antigen or other proteins in a yeast vehicle of thepresent invention is accomplished using techniques known to thoseskilled in the art. Briefly, a nucleic acid molecule encoding at leastone desired antigen or other protein is inserted into an expressionvector in such a manner that the nucleic acid molecule is operativelylinked to a transcription control sequence in order to be capable ofeffecting either constitutive or regulated expression of the nucleicacid molecule when transformed into a host yeast cell. Nucleic acidmolecules encoding one or more antigens and/or other proteins can be inone or more expression vectors operatively linked to one or moreexpression control sequences. Particularly important expression controlsequences are those which control transcription initiation, such aspromoter and upstream activation sequences. Promoters suitable for usein yeast have been described above.

Transfection of a nucleic acid molecule into a cell (e.g., yeast cell)according to the present invention can be accomplished by any method bywhich a nucleic acid molecule can be introduced into the cell andincludes, but is not limited to, diffusion, active transport, bathsonication, electroporation, microinjection, lipofection, adsorption,and protoplast fusion. Transfected nucleic acid molecules can beintegrated into a yeast chromosome or maintained on extrachromosomalvectors using techniques known to those skilled in the art. Examples ofyeast vehicles carrying such nucleic acid molecules are disclosed indetail herein. As discussed above, yeast cytoplast, yeast ghost, andyeast membrane particles or cell wall preparations can also be producedrecombinantly by transfecting intact yeast microorganisms or yeastspheroplasts with desired nucleic acid molecules, producing the antigentherein, and then further manipulating the microorganisms orspheroplasts using techniques known to those skilled in the art toproduce cytoplast, ghost or subcellular yeast membrane extract orfractions thereof containing desired antigens or other proteins.

Effective conditions for the production of recombinant yeast vehiclesand expression of the antigen and/or other protein by the yeast vehicleinclude an effective medium in which a yeast strain can be cultured. Aneffective medium is typically an aqueous medium comprising assimilablecarbohydrate, nitrogen and phosphate sources, as well as appropriatesalts, minerals, metals and other nutrients, such as vitamins and growthfactors. The medium may comprise complex nutrients or may be a definedminimal medium. Yeast strains of the present invention can be culturedin a variety of containers, including, but not limited to, bioreactors,Erlenmeyer flasks, test tubes, microtiter dishes, and Petri plates.Culturing is carried out at a temperature, pH and oxygen contentappropriate for the yeast strain. Such culturing conditions are wellwithin the expertise of one of ordinary skill in the art (see, forexample, Guthrie et al. (eds.), Methods in Enzymology, vol. 194,Academic Press, San Diego (1991)). For example, under one protocol,liquid cultures containing a suitable medium can be inoculated usingcultures obtained from starter plates and/or starter cultures ofyeast-based MUC1 immunotherapy compositions, and are grown forapproximately 20 h at 30° C., with agitation at 250 rpm. Primarycultures can then be expanded into larger cultures as desired. Proteinexpression from vectors with which the yeast were transformed (e.g.,MUC1 expression) may be constitutive if the promoter utilized is aconstitutive promoter, or may be induced by addition of the appropriateinduction conditions for the promoter if the promoter utilized is aninducible promoter (e.g., copper sulfate in the case of the CUP1promoter). In the case of an inducible promoter, induction of proteinexpression may be initiated after the culture has grown to a suitablecell density, which may be at about 0.2 YU/ml or higher densities.

One non-limiting example of a medium suitable for the culture of ayeast-based immunotherapy composition of the invention is U2 medium. U2medium comprises the following components: 15 g/L of glucose, 6.7 g/L ofYeast nitrogen base containing ammonium sulfate, and 0.04 mg/mL each ofhistidine, tryptophan, and adenine, and 0.06 mg/ml of leucine. Anothernon-limiting example of a medium suitable for the culture of yeast-basedimmunotherapy composition of the invention is UL2 medium. UL2 mediumcomprises the following components: 15 g/L of glucose, 6.7 g/L of Yeastnitrogen base containing ammonium sulfate, and 0.04 mg/mL each ofhistidine, tryptophan, and adenine.

In some embodiments of the invention, the yeast are grown under neutralpH conditions (sometimes also referred to as “DEC” or “Dec” conditions).As used herein, the general use of the term “neutral pH” refers to a pHrange between about pH 5.5 and about pH 8, and in one aspect, betweenabout pH 6 and about 8. One of skill the art will appreciate that minorfluctuations (e.g., tenths or hundredths) can occur when measuring witha pH meter. As such, the use of neutral pH to grow yeast cells meansthat the yeast cells are grown in neutral pH for the majority of thetime that they are in culture. In one embodiment, yeast are grown in amedium maintained at a pH level of at least 5.5 (i.e., the pH of theculture medium is not allowed to drop below pH 5.5). In another aspect,yeast are grown at a pH level maintained at about 6, 6.5, 7, 7.5, or 8.In one aspect, neutral pH is maintained by using a suitable buffer tocreate a buffered culture or growth medium. The use of a neutral pH inculturing yeast promotes several biological effects that are desirablecharacteristics for using the yeast as vehicles for immunomodulation.For example, culturing the yeast in neutral pH allows for good growth ofthe yeast without negative effect on the cell generation time (e.g.,slowing of doubling time). The yeast can continue to grow to highdensities without losing their cell wall pliability. The use of aneutral pH allows for the production of yeast with pliable cell wallsand/or yeast that are more sensitive to cell wall digesting enzymes(e.g., glucanase) at all harvest densities. This trait is desirablebecause yeast with flexible cell walls can induce different or improvedimmune responses as compared to yeast grown under more acidicconditions, e.g., by promoting the secretion of cytokines by antigenpresenting cells that have phagocytosed the yeast (e.g., TH1-typecytokines including, but not limited to, IFN-γ, interleukin-12 (IL-12),and IL-2, as well as proinflammatory cytokines such as IL-6). Inaddition, greater accessibility to the antigens located in the cell wallis afforded by such culture methods. In another aspect, the use ofneutral pH for some antigens allows for release of the di-sulfide bondedantigen by treatment with dithiothreitol (DTT) that is not possible whensuch an antigen-expressing yeast is cultured in media at lower pH (e.g.,pH 5). In one non-limiting example of the use of neutral pH conditionsto culture yeast for use in the present invention, UL2 medium describedabove is buffered using, for example, 4.2 g/L of Bis-Tris.

In one embodiment, control of the amount of yeast glycosylation is usedto control the expression of antigens by the yeast, particularly on thesurface. The amount of yeast glycosylation can affect the immunogenicityand antigenicity of the antigen, particularly one expressed on thesurface, since sugar moieties tend to be bulky. As such, the existenceof sugar moieties on the surface of yeast and its impact on thethree-dimensional space around the target antigen(s) should beconsidered in the modulation of yeast according to the invention. Anymethod can be used to reduce or increase the amount of glycosylation ofthe yeast, if desired. For example, one could use a yeast mutant strainthat has been selected to have low glycosylation (e.g., mnn1, och1 andmnn9 mutants), or one could eliminate by mutation the glycosylationacceptor sequences on the target antigen. Alternatively, one could useyeast with abbreviated glycosylation patterns, e.g., Pichia. One canalso treat the yeast using methods that reduce or alter theglycosylation.

In one embodiment of the present invention, as an alternative toexpression of an antigen recombinantly in the yeast vehicle, a yeastvehicle is loaded intracellularly with the polypeptide (protein) orpeptide and/or other molecules that serve as an antigen and/or areuseful as immunomodulatory agents or biological response modifiersaccording to the invention. Subsequently, the yeast vehicle, which nowcontains the antigen and/or other proteins intracellularly, can beadministered to an individual or, alternatively, loaded into a carriersuch as a dendritic cell, which may in turn be administered to anindividual. Peptides and proteins can be inserted directly into yeastvehicles of the present invention by techniques known to those skilledin the art, such as by diffusion, active transport, liposome fusion,electroporation, phagocytosis, freeze-thaw cycles, and bath sonication.Yeast vehicles that can be directly loaded with peptides, proteins,carbohydrates, or other molecules include intact yeast, as well asspheroplasts, ghosts or cytoplasts, which can be loaded with antigensand other agents after production. Alternatively, intact yeast can beloaded with the antigen and/or agent, and then spheroplasts, ghosts,cytoplasts, or subcellular particles can be prepared therefrom. Anynumber of antigens and/or other agents can be loaded into a yeastvehicle in this embodiment, from at least 1, 2, 3, 4 or any wholeinteger up to hundreds or thousands of antigens and/or other agents,such as would be provided by the loading of a microorganism or portionsthereof, for example.

In another embodiment of the present invention, an antigen and/or otheragent is physically attached to the yeast vehicle. Physical attachmentof the antigen and/or other agent to the yeast vehicle can beaccomplished by any method suitable in the art, including covalent andnon-covalent association methods which include, but are not limited to,chemically crosslinking the antigen and/or other agent to the outersurface of the yeast vehicle or biologically linking the antigen and/orother agent to the outer surface of the yeast vehicle, such as by usingan antibody or other binding partner. Chemical cross-linking can beachieved, for example, by methods including glutaraldehyde linkage,photoaffinity labeling, treatment with carbodiimides, treatment withchemicals capable of linking di-sulfide bonds, and treatment with othercross-linking chemicals standard in the art. Alternatively, a chemicalcan be contacted with the yeast vehicle that alters the charge of thelipid bilayer of yeast membrane or the composition of the cell wall sothat the outer surface of the yeast is more likely to fuse or bind toantigens and/or other agent having particular charge characteristics.Targeting agents such as antibodies, binding peptides, solublereceptors, and other ligands may also be incorporated into an antigen asa fusion protein or otherwise associated with an antigen for binding ofthe antigen to the yeast vehicle.

When the antigen or other protein is expressed on or physically attachedto the surface of the yeast, spacer arms may, in one aspect, becarefully selected to optimize antigen or other protein expression orcontent on the surface. The size of the spacer arm(s) can affect howmuch of the antigen or other protein is exposed for binding on thesurface of the yeast. Thus, depending on which antigen(s) or otherprotein(s) are being used, one of skill in the art will select a spacerarm that effectuates appropriate spacing for the antigen or otherprotein on the yeast surface. In one embodiment, the spacer arm is ayeast protein of at least 450 amino acids. Spacer arms have beendiscussed in detail above.

In yet another embodiment, the yeast vehicle and the antigen or otherprotein are associated with each other by a more passive, non-specificor non-covalent binding mechanism, such as by gently mixing the yeastvehicle and the antigen or other protein together in a buffer or othersuitable formulation (e.g., admixture).

In one embodiment, intact yeast (with or without expression ofheterologous antigens or other proteins) can be ground up or processedin a manner to produce yeast cell wall preparations, yeast membraneparticles or yeast fragments (i.e., not intact) and the yeast fragmentscan, in some embodiments, be provided with or administered with othercompositions that include antigens (e.g., DNA vaccines, protein subunitvaccines, killed or inactivated pathogens, viral vector vaccines) toenhance immune responses. For example, enzymatic treatment, chemicaltreatment or physical force (e.g., mechanical shearing or sonication)can be used to break up the yeast into parts that are used as anadjuvant.

In one embodiment of the invention, yeast vehicles useful in theinvention include yeast vehicles that have been killed or inactivated.Killing or inactivating of yeast can be accomplished by any of a varietyof suitable methods known in the art. For example, heat inactivation ofyeast is a standard way of inactivating yeast, and one of skill in theart can monitor the structural changes of the target antigen, ifdesired, by standard methods known in the art. Alternatively, othermethods of inactivating the yeast can be used, such as chemical,electrical, radioactive or UV methods. See, for example, the methodologydisclosed in standard yeast culturing textbooks such as Methods ofEnzymology, Vol. 194, Cold Spring Harbor Publishing (1990). Any of theinactivation strategies used should take the secondary, tertiary orquaternary structure of the target antigen into consideration andpreserve such structure as to optimize its immunogenicity.

Yeast vehicles can be formulated into yeast-based immunotherapycompositions or products of the present invention using a number oftechniques known to those skilled in the art. For example, yeastvehicles can be dried by lyophilization. Formulations comprising yeastvehicles can also be prepared by packing yeast in a cake or a tablet,such as is done for yeast used in baking or brewing operations. Inaddition, yeast vehicles can be mixed with a pharmaceutically acceptableexcipient, such as an isotonic buffer that is tolerated by a host orhost cell. Examples of such excipients include water, saline, Ringer'ssolution, dextrose solution, Hank's solution, and other aqueousphysiologically balanced salt solutions. Nonaqueous vehicles, such asfixed oils, sesame oil, ethyl oleate, or triglycerides may also be used.Other useful formulations include suspensions containingviscosity-enhancing agents, such as sodium carboxymethylcellulose,sorbitol, glycerol or dextran. Excipients can also contain minor amountsof additives, such as substances that enhance isotonicity and chemicalstability. Examples of buffers include phosphate buffer, bicarbonatebuffer and Tris buffer, while examples of preservatives includethimerosal, m- or o-cresol, formalin and benzyl alcohol. Standardformulations can either be liquid injectables or solids which can betaken up in a suitable liquid as a suspension or solution for injection.Thus, in a non-liquid formulation, the excipient can comprise, forexample, dextrose, human serum albumin, and/or preservatives to whichsterile water or saline can be added prior to administration.

The peptide, polypeptide, nucleic acid, vector, or cell can be isolated.The term “isolated” as used herein encompasses compounds or compositionsthat have been removed from a biological environment (e.g., a cell,tissue, culture medium, body fluid, etc.) or otherwise increased inpurity to any degree (e.g., isolated from a synthesis medium). Isolatedcompounds and compositions, thus, can be synthetic or naturallyproduced.

The peptide, polypeptide, nucleic acid, vector, or cell can beformulated as a composition (e.g., pharmaceutical composition)comprising the peptide, polypeptide, nucleic acid, vector, or cell and acarrier (e.g., a pharmaceutically or physiologically acceptablecarrier). Furthermore, the peptide, polypeptide, nucleic acid, vector,cell, or composition of the invention can be used in the methodsdescribed herein alone or as part of a pharmaceutical formulation.

The composition (e.g., pharmaceutical composition) can comprise morethan one peptide, polypeptide, nucleic acid, vector, or cell orcomposition of the invention. Vectors and compositions of the inventioncan further include or can be administered with (concurrently,sequentially, or intermittently with) any other agents or compositionsor protocols that are useful for preventing or treating cancer or anycompounds that treat or ameliorate any symptom of cancer, andparticularly cancers associated with MUC1 expression or overexpression.For example, the composition can comprise one or more otherpharmaceutically active agents or drugs. Examples of such otherpharmaceutically active agents or drugs that may be suitable for use inthe pharmaceutical composition include anticancer agents (e.g.,chemotherapeutic or radiotherapeutic agents), antimetabolites, hormones,hormone antagonists, antibiotics, antiviral drugs, antifungal drugs,cyclophosphamide, and combinations thereof. Suitable anticancer agentsinclude, without limitation, alkylating agents, folate antagonists,purine antagonists, pyrimidine antagonists, spindle poisons,topoisomerase inhibitors, apoptosis inducing agents, angiogenesisinhibitors, podophyllotoxins, nitrosoureas, cisplatin, carboplatin,interferon, asparginase, tamoxifen, leuprolide, flutamide, megestrol,mitomycin, bleomycin, doxorubicin, irinotecan, taxol, geldanamycin(e.g., 17-AAG), and various anti-cancer peptides and antibodies known inthe art.

Exemplary alkylating agents include, but are not limited to, nitrogenmustards (e.g., mechlorethamine, cyclophosphamide, melphalan, uracilmustard, or chlorambucil), alkyl sulfonates (e.g., busulfan),nitrosoureas (e.g., carmustine, lomustine, semustine, streptozocin, ordacarbazine). Exemplary antimetabolites include, but are not limited to,folic acid analogs (e.g., methotrexate), pyrimidine analogs (e.g.,5-fluorouracil (5-FU) or cytarabine), and purine analogs (e.g.,mercaptopurine or thioguanine). Exemplary hormones and hormoneantagonists include, but are not limited to, adrenocorticosteroids(e.g., prednisone), progestins (e.g., hydroxyprogesterone caproate,medroxyprogesterone acetate, and magestrol acetate), estrogens (e.g.,diethylstilbestrol and ethinyl estradiol), antiestrogens (e.g.,tamoxifen), and androgens (e.g., testosterone proprionate andfluoxymesterone). Other exemplary agents include, but are not limitedto, vinca alkaloids (e.g., vinblastine, vincristine, or vindesine),epipodophyllotoxins (e.g., etoposide or teniposide), antibiotics (e.g.,dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, ormitocycin C), enzymes (e.g., L-asparaginase), platinum coordinationcomplexes (e.g., cis-diamine-dichloroplatinum II also known ascisplatin), substituted ureas (e.g., hydroxyurea), methyl hydrazinederivatives (e.g., procarbazine), and adrenocortical suppressants (e.g.,mitotane and aminoglutethimide).

Chemotherapeutics that can be concurrently, sequentially orintermittently administered with the vectors and compositions disclosedherein include Adriamycin, Alkeran, Ara-C, Busulfan, CCNU,Carboplatinum, Cisplatinum, Cytoxan, Daunorubicin, DTIC, 5-FU,Fludarabine, Hydrea, Idarubicin, Ifosfamide, Methotrexate, Mithramycin,Mitomycin, Mitoxantrone, Nitrogen Mustard, Taxol (or other taxanes, suchas docetaxel), Velban, Vincristine, VP-16, Gemcitabine (Gemzar),Herceptin, Irinotecan (Camptosar, CPT-11), Leustatin, Navelbine, RituxanSTI-571, Taxotere, Topotecan (Hycamtin), Xeloda (Capecitabine), Zevelin,Enzalutamide (MDV-3100 or XTANDI™), and calcitriol. Exemplaryimmunomodulators and/or cytokines include, but are not limited to,AS-101 (Wyeth-Ayerst Labs.), bropirimine (Upjohn), gamma interferon(Genentech), GM-CSF (granulocyte macrophage colony stimulating factor;Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immuneglobulin (Cutter Biological), IMREG (from Imreg of New Orleans, La.),SK&F 106528, tumor necrosis factor (TNF)-α, and TNF-β.

Other agents, compositions or protocols (e.g., therapeutic protocols)that are useful for the treatment of cancer in conjunction with thepeptides, polypeptides (proteins), nucleic acids, vectors, cells, andcompositions of the invention include, but are not limited to, surgicalresection of a tumor, radiation therapy, allogeneic or autologous stemcell transplantation, T cell adoptive transfer, and/or targeted cancertherapies (e.g., small molecule drugs, biologics, or monoclonal antibodytherapies that specifically target molecules involved in tumor growthand progression, including, but not limited to, selective estrogenreceptor modulators (SERMs), aromatase inhibitors, tyrosine kinaseinhibitors, serine/threonine kinase inhibitors, histone deacetylase(HDAC) inhibitors, retinoid receptor activators, apoptosis stimulators,angiogenesis inhibitors, poly (ADP-ribose) polymerase (PARP) inhibitors,or immunostimulators).

The additional active agent (e.g., chemotherapeutics agent) can beadministered before, concurrently with (including simultaneously),alternating with, sequentially, or after administration with the vectorsand compositions disclosed herein. In certain embodiments, one or more(e.g., 2, 3, 4, or 5) chemotherapeutic agents is administered incombination with the vectors and compositions disclosed herein. Forexample, when given to an individual in conjunction with chemotherapy ora targeted cancer therapy, it may be desirable to administer theyeast-based immunotherapy compositions during the “holiday” betweendoses of chemotherapy or targeted cancer therapy, in order to maximizethe efficacy of the immunotherapy compositions. Surgical resection of atumor may frequently precede administration of a yeast-basedimmunotherapy composition, but additional or primary surgery may occurduring or after administration of a yeast-based immunotherapycomposition.

The additional active agent can be administered alone or in acomposition. The additional active agent can be formulated by inclusionin a vector (e.g., plasmid or viral vector), in liposomes (tecemotide,which is also known as STIMUVAX™, L-BLP25, or BLP25 liposome vaccine),or in nanoparticles (e.g., VERSAMUNE™ nanotechnology).

The carrier can be any of those conventionally used and is limited onlyby physio-chemical considerations, such as solubility and lack ofreactivity with the active compound(s), and by the route ofadministration. The pharmaceutically acceptable carriers describedherein, for example, vehicles, adjuvants, excipients, and diluents, arewell-known to those skilled in the art and are readily available to thepublic. It is preferred that the pharmaceutically acceptable carrier beone which is chemically inert to the active agent(s) and one which hasno detrimental side effects or toxicity under the conditions of use.

The choice of carrier will be determined in part by the particularpeptide, polypeptide, nucleic acid, vector, cell, or composition thereofof the invention and other active agents or drugs used, as well as bythe particular method used to administer the peptide, polypeptide,nucleic acid, vector, cell, or composition thereof.

The composition additionally or alternatively can comprise one or moreimmunostimulatory/regulatory molecules. Any suitableimmunostimulatory/regulatory molecule can be used, such as interleukin(IL)-2, IL-4, IL-6, IL-12, IL-15, IL-15/IL-15Ra, IL-15/IL-15Ra-Fc,interferon (IFN)-γ, tumor necrosis factor (TNF)-α, B7.1, B7.2, ICAM-1,ICAM-2, LFA-1, LFA-2, LFA-3, CD70, CD-72, RANTES, G-CSF, GM-CSF, OX-40L,41 BBL, anti-CTLA-4, IDO inhibitor, anti-PDL1, anti-PD1, andcombinations thereof. Preferably, the composition comprises acombination of B7.1, ICAM-1, and LFA-3 (also referred to as TRICOM). Theone or more immunostimulatory/regulatory molecules can be administeredin the form of a vector (e.g., a recombinant viral vector, such as apoxvirus vector) comprising a nucleic acid encoding one or moreimmunostimulatory/regulatory molecules. For example, the one or moreimmunostimulatory/regulatory molecules (e.g., IL-12) can be administeredin the form of a DNA plasmid with or without chitosan. Alternatively,the one or more immunostimulatory/regulatory molecules can beadministered as a protein (e.g., recombinant protein), such as a protein(e.g., recombinant IL-12) admixed with chitosan. One or moreimmunostimulatory/regulatory molecules also can be administered incombination with, or concurrently with, a yeast-based immunotherapycomposition of the invention.

In one embodiment of the invention, the composition comprises a firstrecombinant vector comprising the nucleic acid encoding the inventivepeptide or polypeptide (protein) and second recombinant vectorcomprising a nucleic acid encoding B7.1, ICAM-1, and LFA-3. In anotherembodiment, the nucleic acid encoding the inventive peptide orpolypeptide (protein) and the nucleic acid encoding B7.1, ICAM-1, andLFA-3 are in the same recombinant vector. The first and/or secondvectors additionally can comprise a nucleic acid encoding another tumorassociated antigen (e.g., CEA), a modified version thereof (e.g.,CEA-6D), or an epitope thereof.

For example, the recombinant vector can be an avipox vector (e.g.,canarypox virus or a fowlpox virus) comprising the nucleic acid encodingthe inventive peptide and nucleic acids encoding a B7-1 polypeptide, anICAM-1 polypeptide, and an LFA-3 polypeptide. Alternatively, therecombinant vector can be an orthopox virus comprising the nucleic acidencoding the inventive peptide and nucleic acids encoding a B7-1polypeptide, an ICAM-1 polypeptide, and an LFA-3 polypeptide.

In another embodiment of the invention, the composition comprises ayeast-based immunotherapy composition as described herein, wherein theyeast-based immunotherapy composition comprises a yeast vehicle and atleast one antigen comprising the inventive peptide or polypeptide.

The invention provides a method of transducing dendritic cells with thepeptide, polypeptide, nucleic acid, vector, cell, or compositionthereof, and optionally immunostimulatory/regulatory molecules, such asfor example, B7-1, ICAM-1 and LFA-3. In one aspect of the invention,dendritic cells transduced with the peptide, polypeptide, nucleic acid,vector, cell, or composition thereof are administered to the hostgenerate an immune response, such as activation of a cytotoxic T cellresponse.

The invention provides methods of treating a subject suffering from orsusceptible to a MUC1-expressing tumor and/or enhancing an immuneresponse against a MUC1-expressing cancer and/or inhibiting a MUC-1expressing cancer. In a first embodiment, the inventive methods compriseadministering a therapeutically effective amount of one or more of thepeptide, polypeptide, nucleic acid, vector, cell, or composition thereofto a subject. The inventive peptide, polypeptide, nucleic acid, vector,cell, or composition thereof can be used to prevent the development of aMUC1-expressing cancer, particularly in an individual at higher risk todevelop such cancer than other individuals, or to treat a patientafflicted with a MUC1-expressing cancer. The inventive peptide,polypeptide, nucleic acid, vector, cell, or composition thereof isuseful for preventing emergence of such cancers, arresting progressionof such cancers or eliminating such cancers. More particularly, theinventive peptide, polypeptide, nucleic acid, vector, cell, orcomposition thereof can be used to prevent, inhibit or delay thedevelopment of MUC1-expressing tumors, and/or to prevent, inhibit ordelay tumor migration and/or tumor invasion of other tissues(metastases) and/or to generally prevent or inhibit progression ofcancer in an individual. The inventive peptide, polypeptide, nucleicacid, vector, cell, or composition thereof can also be used toameliorate at least one symptom of the cancer, such as by reducing tumorburden in the individual; inhibiting tumor growth in the individual;increasing survival of the individual; and/or preventing, inhibiting,reversing or delaying progression of the cancer in the individual. Theinventive peptide, polypeptide, nucleic acid, vector, cell, orcomposition thereof can be used to treat a subject with any stageMUC1-expressing cancer.

In a second embodiment, the inventive methods comprise obtaining (byisolating) dendritic cells from a subject, treating the dendritic cellswith one or more of the therapeutically effective amount of the peptide,polypeptide, nucleic acid, vector, cell, or composition thereof, andadministering the treated dendritic cells to the subject.

In a third embodiment, the inventive methods comprise (a) obtaining(isolating) peripheral blood mononuclear cells (PBMCs) from a subject,(b) isolating dendritic cells from the PBMCs, (c) treating the dendriticcells with one or more of the therapeutically effective amount of thepeptide, polypeptide, nucleic acid, vector, cell, or composition thereofex vivo, (d) activating the PBMCs with the treated dendritic cells exvivo, and (e) administering the activated PBMCs to the subject.

In a fourth embodiment, the inventive methods comprise a method forinhibiting a MUC1-expressing cancer in a subject comprising (a)obtaining (isolating) PBMCs from a subject, (b) isolating dendriticcells from the PBMCs, (c) treating the dendritic cells with one or moreof the therapeutically effective amount of the peptide, polypeptide,nucleic acid, vector, cell, or composition thereof ex vivo, (d)activating the PBMCs with the treated dendritic cells ex vivo, (e)isolating T lymphocytes from the activated PBMCs ex vivo, and (f)administering the isolated T lymphocytes to the subject.

The invention also provides the use of adoptively transferred T cellsstimulated in vitro with one or more of the therapeutically effectiveamount of the peptide, polypeptide, nucleic acid, vector, cell, orcomposition thereof to inhibit a MUC1-expressing cancer in a subject.

Treatment (e.g., inhibiting a MUC-expressing cancer and/or enhancing animmune response against a MUC1-expressing cancer) comprises, but is notlimited to, destroying tumor cells, reducing tumor burden, inhibitingtumor growth, reducing the size of the primary tumor, reducing thenumber of metastatic legions, increasing survival of the individual,delaying, inhibiting, arresting or preventing the onset or developmentof metastatic cancer (such as by delaying, inhibiting, arresting orpreventing the onset of development of tumor migration and/or tumorinvasion of tissues outside of primary cancer and/or other processesassociated with metastatic progression of cancer), delaying or arrestingprimary cancer progression, improving immune responses against thetumor, improving long term memory immune responses against the tumorantigens, and/or improving the general health of the individual. It willbe appreciated that tumor cell death can occur without a substantialdecrease in tumor size due to, for instance, the presence of supportingcells, vascularization, fibrous matrices, etc. Accordingly, whilereduction in tumor size is preferred, it is not required in thetreatment of cancer.

The MUC1-expressing cancer can be any cancer expressing MUC1 including,but not limited to, human carcinomas (such as ovarian, breast, smallintestine, stomach, kidney, bladder, uterus, testicular, pancreatic,colorectal, lung, thyroid, gastric, head and neck, prostate, esophageal,and other cancers of epithelial cell origin), including primary andmetastatic cancers and hematologic malignancies such as lymphomas,leukemias and myelomas (e.g., multiple myeloma, chronic lymphocyticleukemia (CLL), multiple myelogenous lymphoma (MML), acute myeloidleukemia (AML), Epstein-Barr virus (EBV) transformed B cells, Burkitt'sand Hodgkin's lymphomas and some B-cell non-Hodgkin's lymphomas).

The peptide, polypeptide, nucleic acid, vector, cell, or compositionthereof can be administered to the host by any method. For example, thepeptide, polypeptide, or nucleic acid encoding the peptide orpolypeptide (e.g., as a vector) can be introduced into a cell (e.g., ina host) by any of various techniques, such as by contacting the cellwith the peptide, polypeptide, the nucleic acid, or a compositioncomprising the nucleic acid as part of a construct, as described herein,that enables the delivery and expression of the nucleic acid. Specificprotocols for introducing and expressing nucleic acids in cells areknown in the art (see, e.g., Sambrook et al. (eds.), supra; and Ausubelet al., supra).

A yeast-based immunotherapy composition of the invention can beadministered by various acceptable methods, including, but not limitedto, intravenous administration, intraperitoneal administration,intramuscular administration, intranodal administration, intracoronaryadministration, intraarterial administration (e.g., into a carotidartery), subcutaneous administration, transdermal delivery,intratracheal administration, intraarticular administration,intraventricular administration, inhalation (e.g., aerosol),intracranial, intraspinal, intraocular, aural, intranasal, oral,pulmonary administration, impregnation of a catheter, and directinjection into a tissue. In one aspect, routes of administrationinclude: intravenous, intraperitoneal, subcutaneous, intradermal,intranodal, intramuscular, transdermal, inhaled, intranasal, oral,intraocular, intraarticular, intracranial, and intraspinal. Parenteraldelivery can include intradermal, intramuscular, intraperitoneal,intrapleural, intrapulmonary, intravenous, subcutaneous, atrial catheterand venal catheter routes. Aural delivery can include ear drops,intranasal delivery can include nose drops or intranasal injection, andintraocular delivery can include eye drops. Aerosol (inhalation)delivery can also be performed using methods standard in the art (see,for example, Stribling et al., Proc. Natl. Acad. Sci. USA, 189:11277-11281 (1992)). In one aspect, a yeast-based immunotherapeuticcomposition of the invention is administered subcutaneously. In oneaspect, the yeast-based immunotherapeutic composition is administereddirectly into a tumor milieu.

Suitable methods of administering peptides, polypeptides (proteins),nucleic acids, vectors, cells, and compositions to hosts (subjects) areknown in the art. The host (subject or individual) can be any suitablehost, such as a mammal (e.g., a rodent, such as a mouse, rat, hamster,or guinea pig, rabbit, cat, dog, pig, goat, cow, horse, primate, orhuman).

For example, the peptide, polypeptide, nucleic acid, or vector (e.g.,recombinant poxvirus) can be administered to a host by exposure of tumorcells to the peptide, polypeptide, nucleic acid, or vector ex vivo or byinjection of the peptide, polypeptide, nucleic acid, or vector into thehost. The peptide, polypeptide, nucleic acid, vector (e.g., recombinantpoxvirus) or combination of vectors, cell, and composition can bedirectly administered (e.g., locally administered) by direct injectioninto the cancerous lesion or tumor or by topical application (e.g., witha pharmaceutically acceptable carrier).

The peptide, polypeptide, nucleic acid, vector, cell, or compositionthereof can be administered alone or in combination with adjuvants,incorporated into liposomes (as described in, e.g., U.S. Pat. Nos.5,643,599, 5,464,630, 5,059,421, and 4,885,172), incorporated intonanoparticles (e.g., VERSAMUNE™ nanotechnology), administered withcytokines, administered with biological response modifiers (e.g.,interferon, interleukin-2 (IL-2), administered colony-stimulatingfactors (CSF, GM-CSF, and G-CSF), and/or administered other reagents inthe art that are known to enhance immune response.

Examples of suitable adjuvants include alum, aluminum salts, aluminumphosphate, aluminum hydroxide, aluminum silica, calcium phosphate,incomplete Freund's adjuvant, saponins, such as QS21 (an immunologicaladjuvant derived from the bark of the South American tree Quillajasaponaria Molina), monophosphoryl lipid A (MLP-A), and RIBI DETOX™adjuvant.

A particularly preferred adjuvant for use in the invention is thecytokine GM-CSF. GM-CSF has been shown to be an effective vaccineadjuvant because it enhances antigen processing and presentation bydendritic cells. Experimental and clinical studies suggest thatrecombinant GM-CSF can boost host immunity directed at a variety ofimmunogens.

GM-CSF can be administered using a viral vector (e.g., poxvirus vector)or as an isolated protein in a pharmaceutical formulation. GM-CSF can beadministered to the host before, during, or after the initialadministration of the peptide, polypeptide, nucleic acid, vector, cell,or composition thereof to enhance the antigen-specific immune responsein the host. For example, recombinant GM-CSF protein can be administeredto the host on each day of vaccination with the peptide, polypeptide,nucleic acid, vector, cell, or composition thereof and for each of thefollowing 3 days (i.e. a total of 4 days). Any suitable dose of GM-CSFcan be used. For instance, 50-500 μg (e.g., 100 μg, 200 μg, 300 μg, 400μg, and ranges therebetween) of recombinant GM-CSF can be administeredper day. The GM-CSF can be administered by any suitable method (e.g.,subcutaneously) and, preferably, is administered at or near the site ofthe vaccination of a host with the peptide, polypeptide, nucleic acid,vector, cell, or composition thereof.

In one embodiment, the inventive peptide or polypeptide (protein) can beconjugated to helper peptides or to large carrier molecules to enhancethe immunogenicity of the peptide or polypeptide. These moleculesinclude, but are not limited to, influenza peptide, tetanus toxoid,tetanus toxoid CD4 epitope, Pseudomonas exotoxin A, poly-L-lysine, alipid tail, endoplasmic reticulum (ER) signal sequence, and the like.

The inventive peptide or polypeptide (protein) also can be conjugated toan immunoglobulin molecule using art-accepted methods. Theimmunoglobulin molecule can be specific for a surface receptor presenton tumor cells, but absent or in very low amounts on normal cells. Theimmunoglobulin also can be specific for a specific tissue (e.g., breast,ovarian, colon, or prostate tissue). Such a peptide-immunoglobulinconjugate or polypeptide-immunoglobulin conjugate allows for targetingof the peptide to a specific tissue and/or cell.

The peptide, polypeptide, nucleic acid, vector, cell, or compositionthereof is administered to a host (e.g., mammal, such as a human) in anamount effective to generate a MUC1-specific immune response, preferablya cellular immune response. The efficacy of the peptide, polypeptide,nucleic acid, vector, or cell as an immunogen may be determined by invivo or in vitro parameters as are known in the art. These parametersinclude but are not limited to antigen-specific cytotoxicity assays,regression of tumors expressing MUC1 or MUC1 epitopes, inhibition ofcancer cells expressing MUC1 or MUC1 epitopes, production of cytokines,and the like.

Any suitable dose of the peptide, polypeptide, nucleic acid, vector, orcell or composition thereof can be administered to a host. Theappropriate dose will vary depending upon such factors as the host'sage, weight, height, sex, general medical condition, previous medicalhistory, disease progression, and tumor burden and can be determined bya clinician. For example, the peptide can be administered in a dose ofabout 0.05 mg to about 10 mg (e.g., 0.1 mg, 0.5 mg, 1 mg, 2 mg, 3 mg, 4mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, and ranges therebetween) pervaccination of the host (e.g., mammal, such as a human), and preferablyabout 0.1 mg to about 5 mg per vaccination. Several doses (e.g., 1, 2,3, 4, 5, 6, or more) can be provided (e.g., over a period of weeks ormonths). In one embodiment a dose is provided every month for 3 months.

When the vector is a viral vector, a suitable dose can include about1×10⁵ to about 1×10¹² (e.g., 1×10⁶, 1×10⁷, 1×10⁸, 1×10⁹, 1×10¹⁰, 1×10¹¹,and ranges therebetween) plaque forming units (pfus), although a loweror higher dose can be administered to a host. For example, about 2×10⁸pfus can be administered (e.g., in a volume of about 0.5 mL).

The inventive cells (e.g., cytotoxic T cells) can be administered to ahost in a dose of between about 1×10⁵ and 2×10¹¹ (e.g., 1×10⁶, 1×10⁷,1×10⁸, 1×10⁹, 1×10¹⁰, and ranges therebetween) cells per infusion. Thecells can be administered in, for example, one to three (e.g., one, two,or three) infusions. In addition to the administration of the cells, thehost can be administered a biological response modifier, such asinterleukin 2 (IL-2). When the cells to be administered are cytotoxic Tcells, the administration of the cytotoxic T cells can be followed bythe administration of the peptide, polypeptide, nucleic acid, vector, orcomposition thereof in order to prime the cytotoxic T cells to furtherexpand the T cell number in vivo.

In general, a suitable single dose of a yeast-based immunotherapeuticcomposition is a dose that is capable of effectively providing a yeastvehicle and the MUC1 antigen to a given cell type, tissue, or region ofthe patient body in an amount effective to elicit an antigen-specificimmune response against one or more MUC1 antigens or epitopes, whenadministered one or more times over a suitable time period. For example,in one embodiment, a single dose of a Yeast-MUC1 of the presentinvention is from about 1×10⁵ to about 5×10⁷ yeast cell equivalents perkilogram body weight of the organism being administered the composition.One Yeast Unit (YU) is 1×10⁷ yeast cells or yeast cell equivalents. Inone aspect, a single dose of a yeast vehicle of the present invention isfrom about 0.1 YU (1×10⁶ yeast cells or yeast cell equivalents) to about100 YU (1×10⁹ cells) per dose (i.e., per organism), including anyinterim dose, in increments of 0.1×10⁶ cells (i.e., 1.1×10⁶, 1.2×10⁶,1.3×10⁶, etc.). In one embodiment, a suitable dose includes dosesbetween 1 YU and 40 YU and, in one aspect, between 10 YU and 40 YU orbetween 10 YU and 80 YU In one embodiment, the doses are administered atdifferent sites on the individual but during the same dosing period. Forexample, a 40 YU dose may be administered by injecting 10 YU doses tofour different sites on the individual during one dosing period. Theinvention includes administration of an amount of the Yeast-MUC1immunotherapy composition (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20 YU or more) at 1, 2, 3, 4, 5, 6, 7, 8, 9,10, or more different sites on an individual to form a single dose.

When the cells to be administered are dendritic cells, the amount ofdendritic cells administered to the subject will vary depending on thecondition of the subject and should be determined via consideration ofall appropriate factors by the practitioner. Preferably, about 1×10⁶ toabout 1×10¹² (e.g., about 1×10⁷, about 1×10⁸, about 1×10⁹, about 1×10¹⁰,or about 1×10¹¹ including ranges between of any of the cell numbersdescribed herein) dendritic cells are utilized for adult humans. Theseamounts will vary depending on the age, weight, size, condition, sex ofthe subject, the type of tumor to be treated, the route ofadministration, whether the treatment is regional or systemic, and otherfactors. Those skilled in the art should be readily able to deriveappropriate dosages and schedules of administration to suit the specificcircumstance and needs of the subject.

The invention provides a method of generating peptide-specific cytotoxicT lymphocytes in vivo, ex vivo, or in vitro by stimulation oflymphocytes with an effective amount of the inventive peptide,polypeptide, nucleic acid, vector, or cell, alone or in a compositionwith one or more immunostimulatory/regulatory molecules and/or adjuvantsor in a liposome formulation. The lymphocytes can be lymphocytes fromany suitable source, e.g., peripheral blood, tumor tissues, lymph nodes,and effusions, such as pleural fluid or ascites fluid.

The MUC1 peptide specific cytotoxic T lymphocytes are immunoreactivewith MUC1. Preferably, the cytotoxic T lymphocytes inhibit theoccurrence of tumor cells and cancer and inhibit the growth of, or kill,tumor cells expressing MUC1 or epitopes thereof. The cytotoxic Tlymphocytes, in addition to being antigen specific, can be MEW class Irestricted. In one embodiment, the cytotoxic T lymphocytes are MEW classI HLA-A24 restricted. The cytotoxic T lymphocytes preferably have a CD8⁺phenotype.

In one embodiment, lymphocytes are removed from the host and stimulatedex vivo with the peptide, polypeptide, nucleic acid, vector, cell, orcomposition thereof to generate cytotoxic T lymphocytes. The cytotoxic Tlymphocytes can be administered to the host in order to enhance animmune response to cancer, thereby inhibiting the cancer. Accordingly,the invention provides a method of inhibiting cancer in a hostcomprising (a) obtaining lymphocytes (e.g., from the host), (b)stimulating the lymphocytes with the peptide, polypeptide, nucleic acid,vector, cell, or composition thereof to generate cytotoxic Tlymphocytes, and (c) administering the cytotoxic T lymphocytes to thehost, wherein the cancer is inhibited.

In another embodiment, lymphocytes within the host are stimulated byadministration to the host of the peptide, polypeptide, nucleic acid,vector, cell, or composition thereof to generate cytotoxic Tlymphocytes, which cytotoxic T lymphocytes enhance an immune response tocancer, thereby inhibiting the cancer.

The invention includes a prime and boost protocol. In particular, in oneembodiment related to peptides, polypeptides, and vectors of theinvention, the protocol includes an initial “prime” with a compositioncomprising one or more recombinant vectors encoding the inventivepeptide or polypeptide and optionally one or moreimmunostimulatory/regulatory molecules and/or other tumor-associatedantigens (e.g., CEA), modified versions thereof, and immunogenicepitopes thereof, followed by one or preferably multiple “boosts” with acomposition containing the inventive peptide or polypeptide or one ormore poxvirus vectors encoding the inventive peptide or polypeptide andoptionally one or more immunostimulatory/regulatory molecules and/orother tumor-associated antigens (e.g., CEA), modified versions thereof,and immunogenic epitopes thereof.

In this embodiment, the initial priming vaccination can comprise one ormore vectors. In one embodiment, a single vector (e.g., poxvirus vector)is used for delivery of the inventive peptide and one or moreimmunostimulatory/regulatory molecules and/or other tumor-associatedantigens (e.g., CEA), modified versions thereof, and immunogenicepitopes thereof. In another embodiment, two or more vectors (e.g.,poxvirus vectors) comprise the priming vaccination, which areadministered simultaneously in a single injection.

The boosting vaccinations also can comprise one or more vectors (e.g.,poxvirus vectors). In one embodiment, a single vector is used fordelivery of the inventive peptide and the one or moreimmunostimulatory/regulatory molecules and/or other tumor-associatedantigens (e.g., CEA), modified versions thereof, and immunogenicepitopes thereof of the boosting vaccination. In another embodiment, twoor more vectors comprise the boosting vaccination, which areadministered simultaneously in a single injection.

Different vectors (e.g., poxvirus vectors) can be used to provide aheterologous prime/boost protocol using vectors carrying different setsof therapeutic molecules for inoculations at different time intervals.For example, in one heterologous prime/boost combination, a firstorthopox vector composition is used to prime, and a second avipox vectorcomposition is used to boost.

The schedule for administration of the vectors (e.g., poxvirus vectors)typically involves repeated administration of the boosting vector. Theboosting vector can be administered 1-3 times (e.g., 1, 2, or 3 times)at any suitable time period (e.g., every 2-4 weeks) for any suitablelength of time (e.g., 6-12 weeks for a total of at least 5 to 15boosting vaccinations). For example, the primary vaccination cancomprise a recombinant vaccinia or MVA vector followed by multiplebooster vaccinations with an avipox vector. In a particular embodiment,the host receives one vaccination with the priming vector, followedevery 2 weeks thereafter with the boosting vector for 6 boosts, followedby every 4 weeks thereafter with the boosting vector, and continuingwith the boosting vector for a period of time dependent on diseaseprogression.

The present invention also includes the delivery (administration,immunization, vaccination) of a yeast-based immunotherapeuticcomposition of the invention to a subject or individual. Theadministration process can be performed ex vivo or in vivo, but istypically performed in vivo. Suitable routes of administration andsuitable single doses for yeast-based immunotherapeutic compositionshave been described above. Following an initial (original or priming)dose of a yeast-based immunotherapeutic composition, “boosters” or“boosts” of a yeast-based immunotherapeutic composition areadministered, for example, when the immune response against the antigenhas waned or as needed to provide an immune response or induce a memoryresponse against a particular antigen or antigen(s). Boosters can beadministered about 1, 2, 3, 4, 5, 6, 7, or 8 weeks apart, or monthly,bimonthly, quarterly, annually, and/or in a few or several yearincrements after the original administration (the priming dose),depending on the status of the individual being treated and the goal ofthe therapy at the time of administration (e.g., prophylactic, activetreatment, maintenance). In one embodiment, an administration scheduleis one in which doses of yeast-based immunotherapeutic composition isadministered at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more times overa time period of from weeks, to months, to years. In one embodiment, thedoses are administered weekly or biweekly or triweekly or monthly for 1,2, 3, 4, 5, 6, 7, 8, 9, 10 or more doses, followed by weekly, biweekly,triweekly or monthly doses as needed to achieve the desired preventativeor therapeutic treatment for cancer. Additional boosters can then begiven at similar or longer intervals (months or years) as a maintenanceor remission therapy, if desired.

The invention further provides a kit that, in one embodiment, has atleast a first recombinant vector (e.g., poxvirus vector) that hasincorporated into its genome or portion thereof a nucleic acid encodingthe inventive peptide or polypeptide in a pharmaceutically acceptablecarrier. The first recombinant vector (e.g., poxvirus vectors) also cancomprise one or more nucleic acids encoding one or moreimmunostimulatory/regulatory molecules and/or other tumor-associatedantigens (e.g., CEA), modified versions thereof, and immunogenicepitopes thereof. In addition to the first recombinant vector, the kitcan have a second recombinant vector that comprises one or more nucleicacids encoding one or more immunostimulatory/regulatory molecules and/orother tumor-associated antigens (e.g., CEA), modified versions thereof,and immunogenic epitopes thereof in a pharmaceutically acceptablecarrier. The kit further provides containers, injection needles, andinstructions on how to use the kit. In another embodiment, the kitfurther provides an adjuvant such as GM-CSF and/or instructions for useof a commercially available adjuvant with the kit components.

The invention also includes a kit comprising any of the yeast-basedimmunotherapeutic compositions described herein, or any of theindividual components of such compositions described herein. Kits mayinclude additional reagents and written instructions or directions forusing any of the compositions of the invention to prevent or treatcancer associated with or characterized by MUC1 expression oroverexpression.

As discussed above, the peptide, polypeptide, nucleic acid, vector,cell, or composition thereof can be administered to a host by variousroutes including, but not limited to, subcutaneous, intramuscular,intradermal, intraperitoneal, intravenous, and intratumoral. Whenmultiple administrations are given, the administrations can be at one ormore sites in a host and, in the case of yeast-based immunotherapy, asingle dose can be administered by dividing the single dose into equalportions for administration at one, two, three, four or more sites onthe individual.

Administration of the peptide, polypeptide, nucleic acid, vector, cell,or composition thereof can be “prophylactic” or “therapeutic.” Whenprovided prophylactically, the peptide, polypeptide, nucleic acid,vector, cell, or composition thereof is provided in advance of tumorformation, or the detection of the development of MUC1-expressingtumors, with the goal of preventing, inhibiting or delaying thedevelopment of MUC1-expressing tumors; and/or preventing, inhibiting ordelaying metastases of such tumors and/or generally preventing orinhibiting progression of cancer in an individual, and generally toallow or improve the ability of the host's immune system to fightagainst a tumor that the host is susceptible of developing. For example,hosts with hereditary cancer susceptibility are a preferred group ofpatients treated with such prophylactic immunization. The prophylacticadministration of the peptide, polypeptide, nucleic acid, vector, cell,or composition thereof prevents, ameliorates, or delays theMUC1-expressing cancer. When provided therapeutically, the peptide,polypeptide, nucleic acid, vector, cell, or composition thereof isprovided at or after the diagnosis of the MUC1-expressing cancer, withthe goal of ameliorating the cancer, such as by reducing tumor burden inthe individual; inhibiting tumor growth in the individual; increasingsurvival of the individual; and/or preventing, inhibiting, reversing ordelaying progression of the cancer in the individual.

When the host has already been diagnosed with the MUC1-expressing canceror metastatic cancer, the peptide, polypeptide, nucleic acid, vector,cell, or composition thereof can be administered in conjunction withother therapeutic treatments such as chemotherapy, surgical resection ofa tumor, treatment with targeted cancer therapy, allogeneic orautologous stem cell transplantation, T cell adoptive transfer, otherimmunotherapies, and/or radiation.

In a preferred embodiment, the administration of the peptide,polypeptide, nucleic acid, vector, cell, or composition thereof to ahost results in a host cell expressing the inventive peptide andoptionally one or more immunostimulatory/regulatory molecules and/orother tumor-associated antigens (e.g., CEA), modified versions thereof,and immunogenic epitopes thereof that were co-administered. Theinventive peptide (i.e., MUC1 antigen) can be expressed at the cellsurface of the infected host cell. The one or moreimmunostimulatory/regulatory molecules and/or other tumor-associatedantigens (e.g., CEA), modified versions thereof, and immunogenicepitopes thereof can be expressed at the cell surface or may be activelysecreted by the host cell. The expression of both the MUC1 antigen andthe immunostimulatory/regulatory molecule provides the necessary MEWrestricted peptide to specific T cells and the appropriate signal to theT cells to aid in antigen recognition and proliferation or clonalexpansion of antigen specific T cells. The overall result is anupregulation of the immune system. Preferably, the upregulation of theimmune response is an increase in antigen specific T-helper lymphocytesand/or cytotoxic lymphocytes, which are able to kill or inhibit thegrowth of a cancer (e.g., breast cancer, ovarian cancer, colon cancer,lung cancer, thyroid cancer, gastric cancer, head and neck cancer, orprostate cancer) cell.

There are a variety of suitable formulations of the pharmaceuticalcomposition for the inventive methods. The following formulations forparenteral, subcutaneous, intravenous, intramuscular, andintraperitoneal administration are exemplary and are in no way limiting.One skilled in the art will appreciate that these routes ofadministering the peptide, polypeptide, nucleic acid, vector, cell, orcomposition of the invention are known, and, although more than oneroute can be used to administer a particular compound, a particularroute can provide a more immediate and more effective response thananother route.

Injectable formulations are among those formulations that are preferredin accordance with the present invention. The requirements for effectivepharmaceutical carriers for injectable compositions are well-known tothose of ordinary skill in the art (see, e.g., Pharmaceutics andPharmacy Practice, J.B. Lippincott Company, Philadelphia, Pa., Bankerand Chalmers, eds., pages 238-250 (1982), and ASHP Handbook onInjectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).

Formulations suitable for parenteral administration include aqueous andnon-aqueous, isotonic sterile injection solutions, which can containanti-oxidants, buffers, bacteriostats, and solutes that render theformulation isotonic with the blood of the intended recipient, andaqueous and non-aqueous sterile suspensions that can include suspendingagents, solubilizers, thickening agents, stabilizers, and preservatives.The peptide, polypeptide, nucleic acid, vector, cell, or compositionthereof can be administered in a physiologically acceptable diluent in apharmaceutical carrier, such as a sterile liquid or mixture of liquids,including water, saline, aqueous dextrose and related sugar solutions,an alcohol, such as ethanol, isopropanol, or hexadecyl alcohol, glycols,such as propylene glycol or polyethylene glycol, dimethylsulfoxide,glycerol ketals, such as 2,2-dimethyl-1,3-dioxolane-4-methanol, ethers,such as poly(ethylene glycol) 400, an oil, a fatty acid, a fatty acidester or glyceride, or an acetylated fatty acid glyceride with orwithout the addition of a pharmaceutically acceptable surfactant, suchas a soap or a detergent, suspending agent, such as pectin, carbomers,methylcellulose, hydroxypropylmethylcellulose, orcarboxymethylcellulose, or emulsifying agents and other pharmaceuticaladjuvants.

Oils, which can be used in parenteral formulations, include petroleum,animal, vegetable, and synthetic oils. Specific examples of oils includepeanut, soybean, sesame, cottonseed, corn, olive, petrolatum, andmineral. Suitable fatty acids for use in parenteral formulations includeoleic acid, stearic acid, and isostearic acid. Ethyl oleate andisopropyl myristate are examples of suitable fatty acid esters.

Suitable soaps for use in parenteral formulations include fatty alkalimetal, ammonium, and triethanolamine salts, and suitable detergentsinclude (a) cationic detergents such as, for example, dimethyl dialkylammonium halides, and alkyl pyridinium halides, (b) anionic detergentssuch as, for example, alkyl, aryl, and olefin sulfonates, alkyl, olefin,ether, and monoglyceride sulfates, and sulfosuccinates, (c) nonionicdetergents such as, for example, fatty amine oxides, fatty acidalkanolamides, and polyoxyethylenepolypropylene copolymers, (d)amphoteric detergents such as, for example, alkyl-b-aminopropionates,and 2-alkyl-imidazoline quaternary ammonium salts, and (e) mixturesthereof.

Preservatives and buffers may be used. In order to minimize or eliminateirritation at the site of injection, such compositions may contain oneor more nonionic surfactants having a hydrophile-lipophile balance (HLB)of from about 12 to about 17. The quantity of surfactant in suchformulations will typically range from about 5% to about 15% by weight.Suitable surfactants include polyethylene sorbitan fatty acid esters,such as sorbitan monooleate and the high molecular weight adducts ofethylene oxide with a hydrophobic base, formed by the condensation ofpropylene oxide with propylene glycol.

The parenteral formulations can be presented in unit-dose or multi-dosesealed containers, such as ampoules and vials, and can be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid excipient, for example, water, for injections,immediately prior to use. Extemporaneous injection solutions andsuspensions can be prepared from sterile powders, granules, and tablets.

Yeast-based immunotherapeutic compositions of the invention are mosttypically administered without adjuvant or other carriers and as aninjectable formulation of the yeast-based composition in a simplepharmaceutically acceptable excipient, such as PBS or other buffer.

The following example further illustrates the invention but, of course,should not be construed as in any way limiting its scope.

Example 1

This example describes the analysis of HLA-A24 MUC1-C agonist epitopes.

I. Materials and Methods

Patients—PBMCs were used from two patients with prostate cancer enrolledin a previously described clinical trial of PSA-TRICOM vaccine incombination with ipilimumab (Madan et al., Lancet Oncol., 13: 501-8(2012)). An institutional review board of the National Institutes ofHealth (NIH) Clinical Center had approved the procedures, and informedconsent was obtained in accordance with the Declaration of Helsinki.

Peptides—The MUC1 amino acid sequence was scanned for matches toconsensus motifs for HLA-A24 binding peptides. The computer algorithmdeveloped by Parker et al. to rank potential MHC-binding peptidesaccording to the predicted one-half-time dissociation of peptide/MHCcomplexes was used (Parker et al., J. Immunol., 152: 163-75 (1994)).American Peptide Company (Sunnyvale, Calif.) synthesized 9-mer and10-mer peptide analogues from the MUC1-C region of MUC1 with singleamino acid substitutions in order to increase binding affinity (Table1). The purity of the peptides was >90%.

TABLE 1 MUC1 HLA-A24 binding peptides and potential agonists withpredicted binding and T2-cell binding assay. Predicted Peptide PositionSequence{circumflex over ( )} Binding* C6 462-471 TYHPMSEYPT 6 (SEQ IDNO: 3) C6A KYHPMSEYAL 480 (SEQ ID NO: 1) C7 502-510 SYTNPAVAA 5 (SEQ IDNO: 4) C7A KYTNPAVAL 400 (SEQ ID NO: 2) {circumflex over ( )}Amino acidsthat were changed to generate an agonist epitope are in bold. *Predictedbinding on the basis of reported motif (Parker et al., supra); scoreestimate of half time of disassociation of a molecule containing thissequence.

Affinity and avidity assays—Despite numerous attempts to establishbinding assays for HLA-A24 peptides using T2-A24 cells, reliable assayscould not be established. Therefore, these peptides were evaluated basedsolely on the ability to lyse cells pulsed with the correspondingpeptide and tumor cells expressing the native peptide.

Establishment of T-cell lines—A modified version of the protocoldescribed by Tsang et al., J. Natl. Cancer Inst., 87: 982-90 (1995), wasused to generate MUC1-specific CTLs. Irradiated autologous DCs werepulsed with 20 μg/mL of peptide for 2 hours, and then PBMCs were addedat a 10:1 ratio. After 3 days, human IL-2 (20 Cetus units/mL) was added.Cells were restimulated every 7 days. After the third in vitrostimulation, cells were restimulated using autologous Epstein-Barr virustransformed B cells as antigen presenting cells at a ratio of 2:1, andmaintained in medium containing IL-7 (10 ng/mL) and IL-15 (5 ng/mL).

Detection of cytokines—Autologous B cells pulsed with peptides atdifferent concentrations (25, 12.5, 6.25 and 3, 13, and 1.56 μg/ml) wereincubated with MUC1-specific T-cell lines at a 2:1 ratio for 24 hours.The supernatants were analyzed for IFN-γ by ELISA (Invitrogen,Frederick, Md.).

Tumor cell cultures—The pancreatic carcinoma cell line ASPC-1(HLA-A3^(neg), HLA-A24^(neg), MUC1⁺), colon cancer cell line SW620(HLA-A24⁺, MUC1⁺), and prostate cancer cell line PC3 (HLA-A24⁺, MUC1⁺)were purchased from American Type Culture Collection (Manassas, Va.).All cell cultures were free of mycoplasma and maintained in completemedium (RPMI 1640 supplemented with 10% fetal calf serum, 100 U/mLpenicillin, 100 μg/mL streptomycin, and 2 mM L-glutamine) (Mediatech,Herndon, Va.). K562-A2.1 cells were obtained from Dr. C. Britten(Johannes Gutenberg University, Mainz, Germany), and maintained incomplete medium supplemented with 0.5 mg/mL of G418 (Mediatech,Manassas, Va.).

Cytotoxicity assay, cold target inhibition and antibody blocking oftumor cell lysis—To determine T-cell-mediated killing, a 16-hour¹¹¹Indium release assay was used (Tsang et al., J. Natl. Cancer Inst.,87: 982-90 (1995)). 2×10⁶ target cells were labeled with 60 μCi ¹¹¹Inoxide (GE Health Care, Vienna, Va.) at 37° C. for 20 minutes, and usedat 3000 cells/well in 96-well round-bottom culture plates. T-cells wereadded at different ratios. All assays were performed in RPMI mediumsubstituted with 10% human AB serum (Omega Scientific, Tarzana, Calif.),glutamine and antibiotics (Mediatech, Manassas, Va.). Spontaneousrelease was determined by incubating target cells with medium alone, andcomplete lysis was determined by incubation with 2.5% Triton X-100.Lysis was calculated using the formula:

${{Lysis}(\%)} = {\frac{{{observed}{release}({cpm})} - {{spontaneous}{release}({cpm})}}{{{complete}{release}({cpm})} - {{spontaneous}{release}({cpm})}} \times 100}$A cold target inhibition assay was performed by adding K562-A2.1 orK562-A3 cells, with or without prior pulsing with the correspondingpeptide, at a ratio of 1:10 to the wells (Tsang et al., J. Natl. CancerInst., 87: 982-90 (1995)). Antibody blocking was performed bypre-incubating tumor cells with 10 μg/ml of anti-HLA-A24 antibody orisotype control antibody (UPC10).

II. Analysis

The algorithm for HLA-A24 class I binding peptides in the MUC1-C regionrevealed no potential A24 binders. Changes in anchor residues revealedthe potential for three HLA-A24 agonists. Studies were performed withtwo of these agonists (C6A and C7A, Table 1). The third potentialagonist is not described since a T-cell line generated with the thirdpotential agonist did not lyse tumor cells.

Attempts to generate T-cell lines with the native peptide designated C6were unsuccessful using PBMCs from two different vaccinated cancerpatients. T-cell lines, however, could be generated from these samepatients using APCs pulsed with the corresponding agonist peptide C6A(SEQ ID NO: 1).

The T-cell line derived from APCs pulsed with the C6A peptide wasevaluated for lysis versus two different MUC1⁺, HLA-A24⁺ tumor celllines (SW620; colon cancer, and PC3; prostate cancer) and the ASPC-1pancreatic cancer cell line (MUC1⁺, HLA-A24^(neg)). Lysis of both of theHLA-A24⁺ cell lines was observed (see Table 2) in contrast to theHLA-A24^(neg) line.

TABLE 2 MUC1 native and agonist epitope-specific T-cell lines lyse tumorcells expressing native MUC1 and HLA-A24. SW620 PC3 ASPC-1 E:T MUC1⁺HLA-MUC1⁺HLA- MUC1+HLA- T-cell Line Ratio A24⁺ A24⁺ A24^(neg) T-C6   25:1 NANA NA 12.5:1 NA NA NA T-C6A   25:1 41.2 35.5 2.4 12.5:1 26.0 22.8 1.9T-C7   25:1 22.2 NA 0   12.5:1 13.7 NA NA T-C7A   25:1 41.9 22.6 3.412.5:1 32.6 NA 2.1 Results are expressed as percent (%) specific lysis.The assays were performed at 2 effector (E)-to-target (T) ratios. NA:not available.

The T-cell line derived with the native C7 peptide grew poorly, butenough cells were available to evaluate this T-cell line in acytotoxicity assay using the colon cancer cell line SW620. As can beseen in Table 3, the T-cell line derived with the agonist C7A peptidelysed SW620 cells more efficiently than the T-cell line derived with thenative C7 peptide. Neither T-cell line lysed the ASPC-1 tumor cell line.The addition of an anti-HLA-A24 antibody greatly reduced the lysis oftumor cells, thereby demonstrating the MHC restriction of the lysis forboth the C6A and C7A specific T-cell lines (Table 3).

TABLE 3 MUC1 HLA-A24 agonist epitope-specific T-cell lines lyse tumorcell lines expressing native MUC1 in an HLA-restricted manner. % Lysisof SW620 % Lysis of PC3 T-cell line Blocking MUC1⁺HLA-A24⁺ MUC1⁺HLA-A24⁺T-C6A — 41.2 22.8 Anti-HLA-A24 14.6 10.2 Isotype Control 37.0 20.1 T-C7A— 22.7 22.6 Anti-HLA-A24 8.6 3.1 Isotype Control 17.9 19.7 Results areexpressed as % specific lysis. The assays were performed at an E:T ratioof 25:1 except the T-C6A lysis of PC3 cells, which was performed at anE:T ratio of 12.5:1.

Stimulation of the T-cell line generated with the C6A agonist peptideproduced high levels (pg/mL/10⁵ cells) of IFN-γ (2,651), GM-CSF(>10,000), IL-8 (>10,000), and TNF-α (372), and low levels (<50) ofIL-2, IL-6, IL-10, and IL-12.

T-cell lines could be generated from the same patient using autologousAPCs pulsed with the native C7 or agonist C7A peptides. Each cell linewas then stimulated for 24 hours with B-cells pulsed with either thenative C7 or agonist C7A peptide, and cytokine levels in the supernatantwere analyzed.

As shown in Table 4, the T-cell line generated with the native peptideproduced more Type I cytokine IFN-γ when simulated with the agonist C7Aversus the native C7 peptide. Additionally, when the T-cell linegenerated with the agonist C7A peptide was stimulated with both nativeand agonist peptides, more IFN-γ, GM-CSF, IL-8, IL-10 and TNF-α wasproduced by stimulation with APCs pulsed with agonist C7A peptide versusthe native C7 peptide (Table 4).

TABLE 4 MUC1 HLA-A24 agonist epitope-specific T-cell lines produce Type1 cytokines upon stimulation. T-cell IFN- GM- line Peptide γ CSF IL-2TNFα IL-8 IL-6 IL-10 T-C6A C6A 3060 1277 3630 1021 11.8 7.6 16.4 T-C7 C7750 237 <2.4 21 6.8 6.4 25 C7A 1279 300 <2.4 30 7.3 7.7 45 T-C7A C7 680215 <2.4 30 112 <2.4 92 C7A 2000 910 <2.4 70 360 40 375 Results areexpressed as pg/mL/2.5 × 10⁵ T cells. For the T-C7 and T-C7Aexperiments, the levels of IL-12p70 and IL-1β were <100 pg/mL for thenative and agonist epitopes.

The results of these studies support the therapeutic usefulness ofagonist epitopes of MUC1-C in the context of the invention describedherein, including the use of peptides alone, on dendritic cells, withclassical or novel adjuvant formulation, or with a range of biologicadjuvants, or cytokines such as IL-12, GM-CSF, or IL-15. These agonistpeptides can also be used to activate T cells in vitro in adoptiveT-cell therapy approaches. The T-cell receptors directed against theseagonist epitopes also can be used in genetically engineered T-celladoptive transfer studies. Longer peptides or the MUC1 protein itselfcontaining the agonist epitopes also can be employed as describedherein. Finally, recombinant vector-based vaccines can be employed,which encode the MUC1 transgene and include the sequences for theseagonist epitopes.

Example 2

This example demonstrates the production of a Yeast-based MUC1 agonistimmunotherapeutic composition comprising SEQ ID NO: 1 and known asGI-6108.

Yeast (Saccharomyces cerevisiae) were engineered to express a human MUC1agonist antigen under the control of the copper-inducible promoter,CUP1, producing a yeast-MUC1 agonist immunotherapy composition. The MUC1agonist antigen comprises the enhancer agonist peptide of SEQ ID NO: 1,and was designed using a full-length wild-type MUC1 antigen havingAccession No. NP_001191214 (SEQ ID NO: 14) although other wild-type MUC1proteins could be utilized to design similar agonists.

Briefly, a fusion protein comprising a MUC1 agonist antigen was producedas a single polypeptide with the following sequence elements fused inframe from N- to C-terminus, represented by SEQ ID NO: 16: (1) an alphafactor leader sequence of SEQ ID NO: 17 (corresponding to positions 1-89of SEQ ID NO:16); (2) a linker sequence of Thr-Ser (corresponding topositions 90-91 of SEQ ID NO: 16); (3) a full-length MUC1 agonistprotein corresponding to a wild-type protein except for the introductionof 15 amino acid agonist substitutions and one inactivating substitution(corresponding to positions 92-566 of SEQ ID NO: 16) and (4) ahexapeptide histidine tag (corresponding to positions 567-572 of SEQ IDNO: 16). SEQ ID NO: 16 is encoded by the nucleotide sequence representedby SEQ ID NO: 15 (codon optimized for yeast expression). The alphaleader sequence (corresponding to positions 1-89 of SEQ ID NO: 16) couldbe substituted with a different N-terminal sequence designed to impartresistance to proteasomal degradation and/or stabilize expression, suchas the peptide represented by SEQ ID NO: 19, or an N-terminal peptidefrom a different yeast alpha leader sequence such as SEQ ID NO: 18, orby a MUC1 signal sequence. The hexahistidine C-terminal tag is optional,and facilitates identification and/or purification of the protein. Ascompared to the wild-type MUC1 protein used as a template, the sequenceof SEQ ID NO: 16 contains the following amino acid substitutions:(substitution positions given with reference to SEQ ID NO: 16 withfurther reference in parentheses to the location of the substitution ina wild-type MUC1 represented by Accession No. NP 001191214 identified asSEQ ID NO: 14): T184L (position 93 in wild-type MUC1), A232Y (position141 in wild-type MUC1), P233L (position 142 in wild-type MUC1), G240V(position 149 in wild-type MUC1), S241Y (position 150 in wild-typeMUC1), T242L (position 151 in wild-type MUC1), A483Y (position 392 inwild-type MUC1), C495A (position 404 in wild-type MUC1), C497V (position406 in wild-type MUC1), T513K (position 422 in wild-type MUC1), P521A(position 430 in wild-type MUC1), T522L (position 431 in wild-typeMUC1), T535L (position 444 in wild-type MUC1), D536F (position 445 inwild-type MUC1), and S551Y (position 460 in wild-type MUC1). Thesubstitution C495A (position 404 in the wild-type MUC1 protein) is theinactivating mutation; the remainder of the substitutions are to produceagonist epitopes. SEQ ID NO: 16 comprises the enhancer agonist peptidereferred to herein as SEQ ID NO: 1. SEQ ID NO: 1 is located at positions513-522 of SEQ ID NO: 16. The yeast-based immunotherapy compositioncomprising the whole Saccharomyces cerevisiae yeast expressing thefusion protein of SEQ ID NO: 16 is referred to herein as GI-6108.

A plasmid containing MUC1 agonist antigen for GI-6108 was transfectedinto W303α yeast and transformants were selected after 3 days of growthat 30° C. on uridine dropout agar (UDA). Single colonies werere-streaked onto uridine and leucine dropout agar (ULDA) plates andincubated at 30° C. for an additional 4 days to select for cells withelevated plasmid copy number.

A single colony of GI-6108 was removed from the ULDA plate and used toinoculate 25 mL of UL2 liquid medium (starter culture). pH buffered UL2medium containing 4.2 g/L of Bis-Tris (BT-UL2) also was inoculated withGI-6108 to evaluate this yeast-based immunotherapeutic produced underneutral pH manufacturing conditions (the resulting yeast referred toherein as “GI-6108-DEC”). Culturing in pH buffered UL2 medium exposesβ-glucans on the yeast cell wall and is believed to modify the cellularimmune responses induced by the yeast as a result of modifying theinteractions with dectin receptors on antigen presenting cells.Accordingly, GI-6108 yeast are structurally and functionally differentfrom GI-6108-DEC yeast. The starter cultures were incubated with shakingat 30° C. to a density of ˜3 YU/mL, and then used to inoculate anintermediate culture to 0.3 YU/mL. The intermediate cultures were grownto a density of 3 YU/mL, and then used to inoculate final cultures to adensity of 0.04 YU/mL. The final cultures were grown to a density of 3YU/mL, and then treated with 0.5 mM copper sulfate for 3 h at 30° C. toinduce MUC1 agonist antigen expression.

The induced cells were washed once with PBS, heat killed at 56° C. for 1h, and then thrice washed in PBS. Total protein content of the heatkilled cells was measured by Amidoschwarz assay and the agonist antigencontent was measured by Western blot, with a monoclonal antibodyrecognizing a C-terminal hexahistidine epitope tag. Antigen quantity wasdetermined by interpolation against a standard curve comprised of histagged HCV NS3 protein.

Results showed that the GI-6108 yeast expressed the antigen well in theUL2 medium, and antigen content for GI-6108 was estimated to beapproximately 2531 Ng/YU (data not shown). Expression of antigen byGI-6108-DEC yeast (i.e., GI-6108 grown in BT-UL2 medium, neutral pHconditions) was too low to result in accurate quantification by Westernblot (data not shown). Nonetheless, both GI-6108 and GI-6108-DEC wereused in the experiments described in Example 3.

Example 3

This example demonstrates that yeast-MUC1 immunotherapy compositions ofthe invention known as GI-6108 and GI-6108-DEC can activateMUC1-specific T cells.

T cell lines—T-3-P93L is a MUC-1 specific T cell line that specificallyrecognizes the MUC1 agonist peptide, denoted P93L, in the context ofHLA-A2. P93L is a peptide spanning positions 92-101 of a full-lengthMUC1-C protein (e.g., ATWGQDVTSV, which corresponds to positions 92-101of SEQ ID NO: 14) except that the threonine at position 2 of thispeptide (position 93 of positions 92-101 of SEQ ID NO: 14) issubstituted with a leucine, thereby creating an agonist peptide. P93Lbinds to HLA-A2 at higher levels than the native (wild-type) peptide,and is a better inducer of MUC1-specific T cells than the native peptide(higher production of TH1 cytokines) (see U.S. Patent ApplicationPublication No. 2008/0063653). The T cell line T-3-P93L can specificallylyse HLA-A2-positive, MUC1-positive tumor targets in vitro. This T cellline is specific for a portion of MUC1 that is within the MUC1-Nsubunit.

C1A T cell is a MUC-1 specific T cell line that specifically recognizesthe MUC1 agonist peptide, denoted C1A, in the context of HLA-A2. C1A isa peptide spanning positions 392-401 of a full-length MUC1 protein(e.g., ALAIVYLIAL, which corresponds to positions 392-401 of SEQ ID NO:14) except that the alanine at position 1 of this peptide (position 392of SEQ ID NO: 14) is substituted with a tyrosine, thereby creating anagonist peptide.

C2A T cell is a MUC-1 specific T cell line that specifically recognizesthe MUC1 agonist peptide, denoted C2A, in the context of HLA-A2. C2A isa peptide spanning positions 397-406 of a full-length MUC1 protein(e.g., YLIALAVCQC; which corresponds to positions 397-406 of SEQ ID NO:14) except that the cysteine at position 10 of this peptide (position406 of SEQ ID NO: 14) is substituted with a valine, thereby creating anagonist peptide.

C3A T cell is a MUC-1 specific T cell line that specifically recognizesthe MUC1 agonist peptide, denoted C3A, in the context of HLA-A2. C3A isa peptide spanning positions 460-468 of a full-length MUC1 protein(e.g., SLSYTNPAV, which corresponds to positions 460-468 of SEQ ID NO:14) except that the serine at position 1 of this peptide (position 460in SEQ ID NO: 14) is substituted with a tyrosine, thereby creating anagonist peptide.

V1A T cell is a MUC-1 specific T cell line that specifically recognizesthe MUC1 agonist peptide, denoted VNTR-3, in the context of HLA-A2. V1Ais a peptide spanning positions 150-158 of a full-length MUC1 protein(e.g., STAPPAHGV, which corresponds to positions 150-158 of SEQ ID NO:14) except that the serine at position 1 of this peptide (position 150of SEQ ID NO: 14) is substituted with a tyrosine, and the threonine atposition 2 of this peptide (position 151 of SEQ ID NO: 14) issubstituted with a leucine, thereby creating an agonist peptide.

V2A T cell is a MUC-1 specific T cell line that specifically recognizesthe MUC1 agonist peptide, denoted VNTR-5, in the context of HLA-A2. V2Ais a peptide spanning positions 141-149 of a full-length MUC1 protein(e.g., APDTRPAPG, which corresponds to positions 141-149 of SEQ ID NO:14) except that the alanine at position 1 of this peptide (position 141of SEQ ID NO: 14) is substituted with a tyrosine, and the proline atposition 2 of this peptide (position 142 of SEQ ID NO: 14) issubstituted with a leucine, thereby creating an agonist peptide.

C5A T cell is a MUC-1 specific T cell line that specifically recognizesthe MUC1 agonist peptide, denoted CSA, in the context of HLA-A3. C5A isa peptide spanning positions 443-451 of a full-length MUC1 protein(e.g., STDRSPYEK, which corresponds to positions 443-451 of SEQ ID NO:14) except that the threonine at position 2 of this peptide (position444 of SEQ ID NO: 14) is substituted with a leucine, and the aspartateat position 3 of this peptide (position 445 of SEQ ID NO: 14) issubstituted with a phenylalanine, thereby creating an agonist peptide.

C6A T cell is a MUC-1 specific T cell line that specifically recognizesthe MUC1 agonist peptide, denoted C6A, in the context of HLA-A24. C6A isa peptide spanning positions 422-431 of a full-length MUC1 protein(e.g., TYHPMSEYPT; which corresponds to positions 422-431 of SEQ ID NO:14) except that the threonine at position 1 of this peptide (position422 of SEQ ID NO: 14) is substituted with a tyrosine, the proline atposition 9 of this peptide (position 430 of SEQ ID NO: 14) issubstituted with an alanine, and the threonine at position 10 of thispeptide (position 431 of SEQ ID NO:14) is substituted with a leucine,thereby creating an agonist peptide. This T cell line also is describedin Example 1.

A modified version of the protocol described by Tsang et al., J. Natl.Cancer Inst., 87: 982-90 (1995), was used to generate MUC1-specificCTLs. Irradiated autologous DCs were pulsed with 20 μg/mL of peptide for2 hours, and then PBMCs were added at a 10:1 ratio. After 3 days, humanIL-2 (20 Cetus units/mL) was added. Cells were restimulated every 7days. After the third in vitro stimulation (IVS), cells wererestimulated using autologous Epstein-Barr virus transformed B cells asantigen presenting cells at a ratio of 2:1, and maintained in mediumcontaining IL-7 (10 ng/mL) and IL-15 (5 ng/mL).

In a first experiment, dendritic cells (DCs) from a normal HLA-A2 humandonor were cultured for 48 hours with: (1) medium alone (Medium); (2)GI-6106-DEC yeast (a positive control yeast-MUC1 immunotherapeuticcomposition grown under neutral pH conditions, previously described inPCT Publication No. WO 2013/024972); (3) GI-6108 yeast (a yeast-MUC1immunotherapeutic composition of the invention described in Example 2expressing a MUC1 antigen comprising HLA-A2, HLA-A3 and HLA-A24 agonistepitopes); (4) GI-6108-DEC (a yeast-MUC1 immunotherapeutic compositionof the invention expressing a MUC1 antigen comprising HLA-A2, HLA-A3 andHLA-A24 agonist epitopes that was grown under neutral pH conditions alsoas described in Example 2); and (5) GI-Vec (Yeast Control), a yeastcomprising an empty vector (no MUC1 antigen insert). Treated DCs thenwere used as antigen presenting cells (APCs) to evaluate their abilityto stimulate the MUC1-specific, HLA-A2-restricted T cell lines P93L,C1A, C2A, C3A, V1A and V2A (T cell:DC ratio=10:1). A “no T cell” controlwas also included for each set of DCs. 24 hour culture supernatants werecollected and screened for the secretion of interferon-γ (IFN-γ). Theresults are shown in Table 5, expressed as the amount of IFN-γ producedby the T cells in pg/ml.

TABLE 5 Production of IFN-γ by MUC1-specific HLA-A2 T cells stimulatedwith human DC (HLA-A2) treated with yeast-MUC1 agonist constructs(GI-6108 and GI-6108-DEC) VIA V2A DCs P93L CIA C2A C3A (VNTR- (VNTR-treated T T T T 3) 5) No with: cells cells cells cells T cells T cells Tcell Medium <15.6 <15.6 <15.6 <15.6 <15.6 <15.6 <15.6 GI-6106- 1572 1031603 321 266 148 <15.6 DEC (positive control) HLA- A2/A3 GI-6108 1528 501607 272 153 55 <15.6 HLA- A2/A3/A24 GI-6108- 1165 40 1514 147 81 75<15.6 DEC HLA- A2/A3/A24 GI-Vec <15.6 17 <15.6 <15.6 36 28 <15.6 (YeastControl) Ratio DCs: Yeast = 1:10. Results are expressed in pg/ml 2 × 10⁴DCs: 2 × 10⁵ T cells in 1 ml

As shown in Table 5, dendritic cells treated with GI-6108, producedunder both standard (GI-6108) and neutral pH conditions (GI-6108-DEC),and which express several different MUC1 agonist epitopes, were able tostimulate MUC1-specific, HLA-A2-restricted T cells to producesignificant amounts of IFN-γ in a manner and at an level similar to thepositive control.

In a second experiment, DCs from a normal HLA-A3 or HLA-A24 human donorwere cultured for 48 hours with: (1) medium alone (Medium); (2)GI-6106-DEC yeast; (3) GI-6108 yeast; (4) GI-6108-DEC; and (5) GI-Vec(Yeast Control. Treated DCs were then used as APCs to evaluate theirability to stimulate the MUC1-specific HLA-A3-restricted T cell line C5Aor the MUC1-specific HLA-A24-restricted T cell line C6A (T cell:DCratio=10:1). A “no T cell” control was also included for each set ofDCs. 24 hour culture supernatants were collected and screened for thesecretion IFN-γ. The results are shown in Table 6, expressed as theamount of IFN-γ produced by the T cells in pg/ml.

TABLE 6 Production of IFN-γ by MUC1-specific T cells stimulated withhuman DC (HLA-A3/HLA-A24) treated with Yeast-MUC1 agonist constructs(GI-6108 and GI-6108-DEC) C5A C6A (P483A) (P-462A) No DCs treated with:HLA-A3 T cell line HLA-A24 T cell line T cell Medium <15.6 <15.6 <15.6GI-6106-DEC 4230 1048 <15.6 (positive control) HLA-A2/A3 GI-6108 36642938 <15.6 HLA-A2/A3/A24 GI-6108-DEC 3211 2590 <15.6 HLA-A2/A3/A24GI-Vec <15.6 <15.6 <15.6 (Yeast Control Ratio DCs:Yeast = 1:10. Resultsare expressed in pg/ml of IFN-γ 2 × 10⁴ DCs: 2 × 10⁵ T cells in 1 ml

As shown in Table 6, dendritic cells treated with GI-6108, producedunder both standard (GI-6108) and neutral pH conditions (GI-6108-DEC),and which express A3 and A24 MUC1 agonist epitopes, were able tostimulate both MUC1-specific, HLA-A3-restricted T cells andMUC1-specific HLA-A24-restricted T cells to produce significant amountsof IFN-γ in a manner and at an level similar to the positive control.

This data indicates that a MUC1-specific HLA-A24 T cell line establishedusing MUC1 HLA-A24 agonist epitope C6A can be activated with human DC(HLA-A24 positive) treated with yeast-MUC1 agonist constructs (GI-6108and GI-6108-DEC) containing HLA-A2/A3/A24 MUC1 agonist epitopes andproduce high levels of IFN-γ. Additionally, this data indicates that theMUC1 HLA-A24 agonist epitope-specific T cell line can be activated bythe native HLA-A24 epitope since GI-6106-DEC vector does not contain theHLA-A24 MUC1 agonist epitope.

Example 4

This example describes a phase 1 clinical trial in subjects withMUC1-positive cancer.

An open-label, dose-escalation phase 1 clinical trial is run using ayeast-MUC1 immunotherapy composition known as GI-6108 described inExample 2 (grown either under standard growth conditions or underneutral pH conditions). 12-24 subjects with a MUC1-positive tumor thatcan be HLA-A2, HLA-A3, or HLA-A24 positive are administered theyeast-MUC1 immunotherapy composition in a sequential dose cohortescalation protocol utilizing dose ranges of 4 YU (1 YU×4 sites), 16 YU(4 YU×4 sites), 40 YU (10 YU×4 sites), and 80 YU (20 YU×4 sites)administered subcutaneously. The yeast-MUC1 immunotherapy isadministered at 2 week intervals for 3 months, and then monthly, or isadministered monthly. An expansion cohort of patients (n=10) at maximumtolerated dose (MTD) or the observed best dose are selected foradditional study. The results monitor safety as a primary endpoint, andas secondary endpoints, antigen-specific T cell responses (e.g.,MUC1-specific CD8⁺ T cells emerging or expanding on treatment) as wellas clinical activity.

GI-6108 is expected to be safe and well-tolerated with no significanttoxicities. In addition, GI-6108 is expected to producetreatment-emergent MUC1-specific T cell responses or an improvement inpre-existing MUC1-specific baseline T cell responses in a statisticallysignificant number of patients. Some patients are also expected to havestabilized disease.

Example 5

This example demonstrates that HLA-A24 agonist epitopes of MUC1-C canactivate MUC1-specific cells.

A MUC1-specific HLA-A24 T cell line established using MUC1 HLA-A24agonist epitope C6A was activated with HLA-A24 positive human DCtransfected with poxvirus (MVA) vectors containing MUC1 HLA-A2/A3 MUC1agonist epitopes (MVA-mBN-CV301). In particular, the human DC weretreated with 10 MOI of either (1) MVA-mBN336 clone 73 or (2) MVA-mBN336clone 77 and high levels of IFN-γ were produced (see Table 7).

TABLE 7 MUC1 HLA-A24 agonist epitope (C6A)-specific T cells can beactivated with human DC (HLA-A24 positive) transfected withMVA-mBN-CV-301 vectors and produce high levels of IFN-γ MUC1 HLA-A24agonist epitope (C6A)-specific T cell DCs treated with: line No T cellMedium 33.0 <15.6 MVA-mBN336 1034 24.6 clone 73 (containing MUC1HLA-A2/A3 agonist epitopes and native HLA-A24 epitope) MVA-mBN336 99622.4 clone 77 (containing MUC1 HLA-A2/A3 agonist epitopes and nativeHLA-A24 epitope) Results are expressed in pg/ml of IFN-γ 2 × 10⁴ DCs: 2× 10⁵ T cells in 1 ml

As shown in Table 7, MUC1 HLA-A24 agonist epitope-specific T cell linescan be activated by the native HLA-A24 epitope since MVA-mBN-CV301vectors do not contain the HLA-A24 MUC1 agonist epitope.

All references, including publications, patent applications, andpatents, cited herein are hereby incorporated by reference to the sameextent as if each reference were individually and specifically indicatedto be incorporated by reference and were set forth in its entiretyherein.

The use of the terms “a” and “an” and “the” and “at least one” andsimilar referents in the context of describing the invention (especiallyin the context of the following claims) are to be construed to coverboth the singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The use of the term “at least one”followed by a list of one or more items (for example, “at least one of Aand B”) is to be construed to mean one item selected from the listeditems (A or B) or any combination of two or more of the listed items (Aand B), unless otherwise indicated herein or clearly contradicted bycontext. The terms “comprising,” “having,” “including,” and “containing”are to be construed as open-ended terms (i.e., meaning “including, butnot limited to,”) unless otherwise noted. Recitation of ranges of valuesherein are merely intended to serve as a shorthand method of referringindividually to each separate value falling within the range, unlessotherwise indicated herein, and each separate value is incorporated intothe specification as if it were individually recited herein. All methodsdescribed herein can be performed in any suitable order unless otherwiseindicated herein or otherwise clearly contradicted by context. The useof any and all examples, or exemplary language (e.g., “such as”)provided herein, is intended merely to better illuminate the inventionand does not pose a limitation on the scope of the invention unlessotherwise claimed. No language in the specification should be construedas indicating any non-claimed element as essential to the practice ofthe invention.

Preferred embodiments of this invention are described herein, includingthe best mode known to the inventors for carrying out the invention.Variations of those preferred embodiments may become apparent to thoseof ordinary skill in the art upon reading the foregoing description. Theinventors expect skilled artisans to employ such variations asappropriate, and the inventors intend for the invention to be practicedotherwise than as specifically described herein. Accordingly, thisinvention includes all modifications and equivalents of the subjectmatter recited in the claims appended hereto as permitted by applicablelaw. Moreover, any combination of the above-described elements in allpossible variations thereof is encompassed by the invention unlessotherwise indicated herein or otherwise clearly contradicted by context.

The invention claimed is:
 1. A yeast-MUC1 immunotherapeutic composition,wherein the immunotherapeutic composition comprises: (a) a yeastvehicle; and (b) a fusion protein comprising at least one MUC1 antigen,wherein the MUC1 antigen has an amino acid sequence that differs fromthe amino acid sequence of a wild-type MUC1 protein comprising SEQ IDNO:14 by at least one amino acid substitution at a sequence position,with respect to the wild-type MUC1 amino acid sequence, selected fromthe group consisting of: T93, A141, P142, G149, S150, and T151, andwherein the fusion protein has been expressed by the yeast vehicle. 2.The Yeast-MUC1 immunotherapeutic composition of claim 1, wherein theyeast vehicle is a whole yeast.
 3. The Yeast-MUC1 immunotherapeuticcomposition of claim 1, wherein the yeast vehicle has beenheat-inactivated.
 4. The Yeast-MUC1 immunotherapeutic composition ofclaim 1, wherein the yeast vehicle is from Saccharomyces cerevisiae. 5.The Yeast-MUC1 immunotherapeutic composition of claim 1, wherein theMUC1 antigen has an amino acid sequence that differs from the amino acidsequence of the wild-type MUC1 protein having SEQ ID NO:14, by at leastone amino acid substitution at a sequence position, with respect to thewild-type MUC1 amino acid sequence, selected from the group consistingof: T93L, A141Y, P142L, G149V, S150Y, and T151L.
 6. The Yeast-MUC1immunotherapeutic composition of claim 1, wherein the immunotherapeuticcomposition has been produced by culturing a whole yeast expressing theMUC1 antigen in a medium that was maintained at a pH level of between5.5 and 8.